Small molecule inhibitors of STAT3 with anti-tumor activity

ABSTRACT

The present invention concerns compounds, compositions containing these compounds, and methods of using these compounds and compositions as inhibitors of Stat3 signaling, Stat3 dimerization, Stat3-DNA binding, Stat5-DNA binding, and/or aberrant cell growth in vitro or in vivo, e.g., as anti-cancer agents for treatment of cancer, such as breast cancer. The compounds of the invention include, but are not limited to, NSC 74859 (S3I-201), NSC 42067, NSC 59263, NSC 75912, NSC 11421, NSC 91529, NSC 263435, and pharmaceutically acceptable salts and analogs of the foregoing. Other non-malignant diseases characterized by proliferation of cells that may be treated using the compounds of the invention, but are not limited to, cirrhosis of the liver; graft rejection; restenosis; and disorders characterized by a proliferation of T cells such as autoimmune diseases, e.g., type 1 diabetes, lupus and multiple sclerosis. The invention further includes an in-vitro screening test for the presence of malignant cells in a mammalian tissue; a method of identifying inhibitors of constitutive Stat3 activation, Stat3-DNA binding, Stat5-DNA binding, and/or Stat3 dimerization; and a method of identifying anti-cancer agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation application of U.S.application Ser. No. 15/468,460, filed Mar. 24, 2017, which is acontinuation application of U.S. application Ser. No. 14/246,522, filedApr. 7, 2014, now U.S. Pat. No. 9,604,923, which is a continuationapplication of U.S. application Ser. No. 12/931,607, filed Feb. 4, 2011,now abandoned, which is a continuation application of U.S. applicationSer. No. 11/805,217, filed May 21, 2007, now U.S. Pat. No. 7,960,434,which claims the benefit of U.S. Provisional Application Ser. No.60/801,750, filed May 19, 2006, each of which is hereby incorporated byreference herein in its entirety, including any figures, tables, nucleicacid sequences, amino acid sequences, and drawings.

GOVERNMENT SUPPORT

This invention was made with government support under CA106439 awardedby the National Institutes of Health and W81XWH-08-2-0101 awarded byUnited States Army Medical Research and Materiel Command. The governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

Signal transduction proteins important in carcinogenesis and cancerprogression present attractive targets for the development of novelanticancer therapeutics. The family of proteins, Signal Transducer andActivator of Transcription (STAT), are activated in response tocytokines and growth factors and promote proliferation, survival, andother biological processes (Bromberg, J. Breast Cancer Res., 2000,2:86-90; Darnell, J. E., Jr. Nat. Rev. Cancer, 2002, 2:740-749; Yu, H.and Jove, R. Nat. Rev. Cancer, 2004, 4:97-105). STATs are activated byphosphorylation of a critical tyrosine residue, which is mediated bygrowth factor receptor tyrosine kinases, Janus kinases or the Src familykinases. Upon tyrosine phosphorylation, dimers of STATs formed betweentwo phosphorylated monomers translocate to the nucleus, bind to specificDNA-response elements in the promoters of target genes, and induce geneexpression. Aberrant activity of one of the family members, Stat3,contributes to carcinogenesis and tumor progression by upregulating geneexpression and promoting dysregulated growth, survival, andangiogenesis, and modulating immune responses (Darnell, J. E., Jr. Nat.Rev. Cancer, 2002, 2:740-749; Yu, H. and Jove, R. Nat. Rev. Cancer,2004, 4:97-105; Bromberg, J. and Darnell, J. E., Jr. Oncogene, 2000,19:2468-2473; Bowman, T. et al. Oncogene, 2000, 19:2474-2488; Turkson,J. and Jove, R. Oncogene, 2000, 19:6613-6626; Buettner, R. et al. Clin.Cancer Res., 2002, 8:945-954; Turkson, J. Expert Opin Ther Targets,2004, 8:409-422; Darnell, J. E. Nat Med., 2005, 11:595-596).

As a critical step in STAT activation (Shuai, K. et al. Cell, 1994,76:821-828), the dimerization between two STAT monomers presents anattractive target to abolish Stat3 DNA-binding and transcriptionalactivity and inhibit Stat3 biological functions (Turkson, J. et al. J.Biol. Chem., 2001, 276:45443-45455; Turkson, J. et al. Mol Cancer Ther,2004, 3:261-269). Stat3 dimerization relies on the reciprocal binding ofthe SH2 domain of one monomer to the pTyr peptide containing APY*LKT(Ala-Pro-pTyr-Leu-Lys-Thr) (SEQ ID NO:1) sequence of the other Stat3monomer. To pursue the development of inhibitors of Stat3 signaling, keystructural information gleaned from the X-ray crystal structure of theStat3(3 homodimer (Becker, S. et al. Nature, 1998, 394:145-151) was usedin the computational modeling and automated docking of small-moleculesinto the SH2 domain of a Stat3 monomer, relative to the bound nativepTyr peptide, in order to identify binders of the Stat3 SH2 domain, andpotentially disruptors of Stat3: Stat3 dimers (Shao, H. et al. J BiolChem., 2004, 279:18967-18973; Song, H. et al. Proc Natl Acad Sci USA,2005, 102:4700-4705).

BRIEF SUMMARY OF THE INVENTION

Structure-based high throughput virtual screening of the National CancerInstitute (NCI) Chemical libraries identified three compounds thatselectively inhibit Stat3 DNA-binding activity in vitro, with IC₅₀values of 65-86 μM, namely NSC 74859, NSC 59263, and NSC 42067. Thehighest scoring compound, NSC 74859 (re-synthesized as a pure sample andnamed S3I-201), selectively inhibits Stat3 DNA-binding activity in vitrowith an IC₅₀ value of 86±33 μM. Furthermore, S3I-201 induces growthinhibition and apoptosis of malignant cells in part by inhibitingconstitutively-active Stat3, and induces human breast tumor regressionin xenograft models. These findings support the therapeutic potential ofS3I-201 and other Stat3 inhibitors against tumors harboring aberrantStat3 activity.

The present invention concerns isolated compounds, compositionscontaining these compounds, and methods of using these compounds andcompositions as inhibitors of Stat3 and/or as inhibitors of aberrantcell growth, e.g., as anti-cancer agents. In one embodiment, thecompound has a structure described by Formula A, B, C, D, E, or F inFIG. 10, 11, or 12A-D, respectively, or a pharmaceutically acceptablesalt or analog thereof. In another embodiment, the compound is NSC 74859(S3I-201; shown in FIG. 7), NSC 59263 (shown in FIG. 8), NSC 42067(shown in FIG. 9), NSC 75912 (shown in FIG. 50), NSC 11421 (shown inFIG. 49), NSC 91529 (shown in FIG. 51), NSC 263435 (shown in FIG. 48),or a pharmaceutically acceptable salt or analog of any of the foregoing.In another embodiment, the compound is an analog of S3I-201 shown inFIGS. 13-47, i.e., a compound selected from the group consisting ofHL2-006-1, HL2-006-2, HL2-006-3, HL2-006-4, HL2-006-5, HL2-011-1,HL2-011-2, HL2-011-3, HL2-011-4, HL2-011-5, BG2069-1, HL2-011-6,HL2-011-7, HL2-005, HL2-003, BG2066, BG2074, BG3004, BG3006A, BG3006B,BG3006D, BG3009, RPM381, RPM384, RPM385, RPM405, RPM411, RPM407, RPM412,RPM408, RPM410, RPM415, RPM416, RPM418, RPM418-A, RPM427, RPM431,RPM432, RPM444, RPM448, RPM445, RPM447, RPM452, RPM202, or apharmaceutically acceptable salt or analog of any of the foregoing. Inanother embodiment, the compound is one listed in Table 4, or apharmaceutically acceptable salt or analog thereof.

One aspect of the invention concerns a method of treating aproliferation disorder in a subject, comprising administering aneffective amount of at least one compound of the invention to thesubject. In one embodiment, the disorder is mediated by cells harboringconstitutively-active Stat3.

Another aspect of the invention concerns a method of suppressing thegrowth of malignant cells, comprising contacting the cells in vitro orin vivo with an effective amount of at least one compound of theinvention. In one embodiment, the malignant cells harborconstitutively-active Stat3.

Another aspect of the invention concerns a method of inducing apoptosisin malignant cells, comprising contacting the cells in vitro or in vivowith an effective amount of at least one compound of the invention. Inone embodiment, the malignant cells harbor constitutively-active Stat3.

Another aspect of the invention concerns a method of inhibitingconstitutive activation of Stat3 in cells, comprising contacting thecells in vitro or in vivo with an effective amount of at least onecompound of the invention.

Another aspect of the invention concerns a method of preventing Stat3dimerization in a mammalian cell, the method comprising contacting thecell in vitro or in vivo with an effective amount of at least compoundof the invention.

Another aspect of the invention concerns a a method of disruptingStat3-DNA binding, the method comprising contacting the Stat3 with aneffective amount of at least one compound of the invention.

Another aspect of the invention concerns a a method of disruptingStat5-DNA binding, the method comprising contacting the Stat5 with aneffective amount of at least one compound of the invention.

Another aspect of the invention concerns an in-vitro screening test forthe presence of malignant cells in a mammalian tissue, the testincluding: obtaining a sample containing viable cells of the tissue;culturing the sample under conditions promoting growth of the viablecells contained therein; treating the cultured sample with a compound ofthe invention; and analyzing the treated sample by a method effective todetermine percent apoptosis of cells as an indicator of presence ofmalignant cells in the sample.

Another aspect of the invention concerns a method of identifyinginhibitors of constitutive Stat3 activation, Stat3-DNA binding,Stat5-DNA binding, and/or Stat3 dimerization, the method comprisingselecting a compound having a structure of Formula A, B, C, D, E, or F(shown in FIGS. 10-12A-D); and determining whether the compound inhibitsconstitutive Stat3 activation, disrupts Stat3-DNA binding, disruptsStat5-DNA binding, prevents (e.g., reduces incidence of) Stat3dimerization, or determining two or more of the foregoing.

Another aspect of the invention concerns a method of identifyinganti-cancer agents, the method comprising selecting a compound having astructure of Formula A, B, C, D, E, or F (shown in FIGS. 10-12A-D); anddetermining whether the compound inhibits the growth of cancer cells invitro or in vivo (e.g., in an animal model).

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Patent and Trademark Officeupon request and payment of the necessary fee.

FIGS. 1A-1B show docking of S3I-201 (NSC 74859) to the SH2 domain ofStat3. FIG. 1A depicts S3I-201 docked to the SH2 domain of Stat3; asolvent accessible surface of the protein (rendered on a 6 Å shell ofresidues surrounding the ligand) is shown. It is color coded accordingto the electrostatic potential. Hydrogen bonding to Lys 591, Ser 611,Ser 613, and Arg 609 is shown by white dashed lines. Carbon atoms ofS3I-201 are shown in green. FIG. 1B depicts S3I-201 docked to the SH2domain of Stat3 along with pTyr peptide. Coloring for pTyr peptide is asfollows: (P: red, Y*: pink; L: orange; K: green).

FIGS. 2A-1, 2A-2, 2B-1, 2B-2, 2C-1, 2C-2, 2D, 2E, 2F, 2G-1, 2G-2, 2G-3,and 2G-4 show the effects of S3I-201 on STATs, Shc, Src, and Erksactivation, and on Lck-SH2 domain-phosphopeptide binding. In FIGS. 2A-1and 2A-2, nuclear extracts containing activated Stat1, Stat3, and/orStat5 proteins were pre-incubated with or without S3I-201 for 30 minutesat room temperature prior to incubation with radiolabeled hSIE probethat binds Stat1 and Stat3 or MGFe probe that binds Stat1 and Stat5, ornuclear extracts were incubated with anti-Stat1, anti-Stat3, oranti-Stat5A antibody for 30 minutes at room temperature prior toincubation with radiolabeled probes, and subjecting to EMSA analysis. InFIG. 2B-1, cell lysates containing activated Stat3 were pre-incubatedwith S3I-201 or without (control, 0.05% DMSO) in the presence or absenceof increasing amount of cell lysates containing inactive Stat3 protein(monomer), inactive Stat1 protein (monomer), inactive Stat5 protein(monomer), or Src protein prior to incubation with radiolabeled hSIEprobe and subjecting to EMSA analysis. FIG. 2B-2 shows SDS-PAGE andWestern blot analysis of cell lysate preparations of equal total proteincontaining inactive Stat3 (monomer), Stat1 (monomer), or Stat5(monomer), or Src protein probing with anti-Stat3, anti-Stat1,anti-Stat5, or anti-Src antibody. FIG. 2C-1 shows SDS-PAGE and Westernblot analysis of Stat3-YFP immunoprecipitates (i.p. YFP, left panels)probing for FLAG-Stat3 (anti-FLAG, upper panel) or Stat3-YFP (anti-YFP,lower panel), or of FLAG-Stat3 immunoprecipitates (i.p. FLAG, rightpanels) probing for Stat3-YFP (anti-YFP, lower panel) or FLAG-Stat3(anti-FLAG, upper panel). FIG. 2C-2 shows SDS-PAGE and Western blotanalysis of whole-cell lysates from FLAG-ST3-transiently-transfectedcells (input) treated with S3I-201 or untreated (control) probing forFLAG. FIG. 2D shows in vitro ELISA for the binding of Lck-SH2-GST to theconjugate biotinylated pTyr-peptide (EPQpYEEIEL (where pY representspTyr)) (SEQ ID NO:2), and effects of increasing concentration ofS3I-201. FIG. 2E shows EMSA analysis of nuclear extract preparationsfrom v-Src-transformed mouse fibroblasts (NIH3T3/v-Src) treated for theindicated times or from the human breast cancer MDA-MB-231, MDA-MB-435and MDA-MB-468 cells treated for 48 hours with 100 μM S3I-201 andincubated with radiolabeled hSIE probe. FIG. 2F shows SDS-PAGE andWestern blot analysis of whole-cell lysates from NIH3T3/v-Srcfibroblasts untreated (control) or treated with S3I-201 (100 μM, 24hours) probing with anti-pTyr705 Stat3 or anti-Stat3 antibody. FIGS.2G-1, 2G-2, 2G-3, and 2G-4 show SDS-PAGE and Western blot analysis ofcell lysates prepared from NIH3T3/v-Src or EGF-stimulated NIH3T3/hEGFRuntreated (control) or treated with S3I-201 (100 μM, 24 hours) probingwith antibodies against pShc (FIG. 2G-1), pErk1/2 (pp42/pp44), Erk1/2(FIG. 2G-2), pSrc (FIG. 2G-3), or β-actin, or anti-phosphoTyr antibody,clone 4G10 (FIG. 2G-4). Positions of STATs:DNA complexes or proteins ingel are labeled; control lanes represent nuclear extracts treated with0.05% DMSO, and nuclear extracts or cell lysates prepared from 0.05%DMSO-treated cells; asterisks (*) indicates supershifted complexes ofSTAT:probe:antibody in the presence of anti-Stat1, anti-Stat3 oranti-Stat5 antibody; values are the mean and S.D. of three independentdeterminations.

FIGS. 3A-3C show that S3I-201 suppresses Stat3-dependent but notStat3-independent transcriptional activity. Luciferase reporteractivities in cytosolic extracts prepared from normal mouse fibroblasts(NIH3T3) transiently co-transfected with the Stat3-dependent (pLucTKS3[FIG. 3A]), or the Stat3-independent (pLucSRE [FIG. 3B] or β-Caseinpromoter-driven Luc [FIG. 3C]) luciferase reporters together with aplasmid expressing the v-Src oncoprotein that activates all threereporters, and untreated (0.05% DMSO, control) or treated with 100 μMS3I-201 for 24. Values are the mean and S.D. of three independentdeterminations.

FIGS. 4A-4G show that S3I-201 inhibits anchorage-dependent and-independent growth only of cells that contain persistently-activeStat3. Normal mouse fibroblasts (NIH3T3) (FIG. 4A) and v-Src transformedcounterparts (NIH3T3/v-Src) (FIG. 4B), as well as the human breastcarcinoma cells (MDA-MB-453 [FIG. 4C]), MDA-MB-435 [FIG. 4D], MDA-MB-231[FIG. 4E], or MDA-MB-468 [FIG. 4F]) were untreated (0.05% DMSO, control)or treated with 100 μM S3I-201 and counted by trypan blue exclusion oneach of four days for viable cell number. In FIG. 4G, v-Src transformedmouse fibroblasts (NIH3T3/v-Src) and their v-Ras transformedcounterparts (NIH3T3/v-Ras) were grown in soft-agar suspension anduntreated (0.05% DMSO, control) or treated with 100 μM S3I-201 every 3days until large colonies were visible, which were enumerated. Valuesare the mean and S.D. of 3-4 independent determinations.

FIGS. 5A-5C show that S3I-201 inhibits Cyclin D1, Bcl-xL and Survivinexpression and induces apoptosis in a Stat3-dependent manner. In FIG.5A, normal NIH3T3 mouse fibroblasts and their v-Src transformedcounterparts (NIH3T3/v-Src), and the human breast carcinoma MDA-MB-453and MDA-MB-435 cell lines were untreated (0.05% DMSO) or treated with100-300 μM S3I-201 for 48 hours and subjected to Annexin V staining andFlow Cytometry. In FIG. 4B, human breast carcinoma MDA-MB-231 cells weretransfected with pcDNA3 (mock), Stat3C or untransfected (Non), or thev-Src transformed mouse fibroblasts (NIH3T3/v-Src) were transfected withpcDNA3 (mock), N-terminus of Stat3 (ST3-NT) or the Stat3 SH2 domain(ST3-SH2) for 4 hours. Twenty four hours after transfection, cells wereuntreated (0.05% DMSO, (−)) or treated (+) with 100 μM S3I-201 for anadditional 24 hours and subjected to Annexin V staining and FlowCytometry. FIG. 5C shows SDS-PAGE and Western blot analysis ofwhole-cell lysates prepared from the v-Src-transformed mouse fibroblasts(NIH3T3/v-Src) or the human breast cancer MDA-MB-231 cells untreated(DMSO, control) or treated with 100 μM S3I-201 for 48 hours probing withanti-Cyclin D1, Bcl-xL and Survivin antibodies. Values are the mean andS.D. of six independent determinations. Western blot data arerepresentative of 3 independent analyses.

FIGS. 6A-6C show tumor growth inhibition by S3I-201. In FIG. 6A, humanbreast (MDA-MB-231) tumor-bearing mice were given S3I-201 (5 mg/kg) i.v.every 2 or every 3 days. Tumor sizes were monitored every 2 to 3 days,converted to tumor volumes, and plotted. In FIG. 6B, upon completion ofstudy 3 days after the last S3I-201 injection, animals were sacrificedand tumor from one control animal (DMSO-treated) or residual tumortissue from S3I-201 treated (T1 and T2) mice were extracted and lysatepreparations of equal total proteins were analyzed for Stat3 activationby incubating with radiolabeled hSIE probe and subjecting to EMSAanalysis (lanes 1 to 3), or lysates from control tumor tissue of equalprotein were pre-incubated with or without increasing concentration ofS3I-201 prior to incubation with radiolabeled hSIE probe and subjectingto EMSA analysis (lanes 1, 4, 5 and 6). In FIG. 6C, lysate preparationsfrom extracted tumor tissue in control or treated (T1 and T2) weresubjected to SDS-PAGE and Western blot analysis for pTyr Stat3 (pYStat3,upper panel) and total Stat3 (lower panel). Values are the mean and S.D.of eight tumor-bearing mice each. Positions of Stat3:DNA complexes areshown.

FIG. 7 shows the chemical structure of NSC-74859 (also referred toherein as S3I-201).

FIG. 8 shows the chemical structure of NSC-59263.

FIG. 9 shows the chemical structure of NSC-42067.

FIG. 10 shows the chemical structure of formula A, encompassing someembodiments of the invention. R, if present ═H or OH; Y═O, NH, NR, CH₂,CHR, or CR₂; and X, if present=a phosphate mimic, e.g., CO₂H, SO₃H,PO₃H, NO₂, CH₂CO₂H, or CF₂CO₂H, CF(CO₂H)₂ tetrazole.

FIG. 11 shows the chemical structure of formula B, encompassing someembodiments of the invention. R, if present ═H or OH; X, if present═phosphate mimic, e.g., CO₂H, SO₃H, PO₃H, NO₂, CH₂CO₂H, or CF₂CO₂H,CF(CO₂H)₂ tetrazole.

FIGS. 12A-12D show the structures of T-shaped molecules (Formulas C, D,E, and F, respectively) that bind in the SH2 binding groove. Thehydrophobic group containing R₂ binds in a STAT3 hydrophobic pocketformed from Ile634, Ile597, Lys591, and Arg595. In FIGS. 12A-12D, theXNH groups have a transposed arrangement (NHX) with respect to oneanother. Referring to FIGS. 12A-12D, R₁ and R₂, if present, can be analiphatic or aromatic group; X, if present ═CO, SO₂, CONH, or alkyl; Z,if present, is alkyl; and phosphate mimic=that shown in FIGS. 10 and 11,e.g., CO₂H, SO₃H, PO₃H, NO₂, CH₂CO₂H, CF₂CO₂H, or CF(CO₂H)₂ tetrazole.Preferably, R₂ is a hydrophobic group or part of a hydrophobic group. Inone embodiment, R₁, if present, is H, alkyl, alkenyl, cycloalkyl,heterocycloalkyl, cylcoalkenyl, heterocycloalkenyl, acyl, and aryl, anyof which may be optionally substituted; and R₂, if present is H, alkyl,alkenyl, cycloalkyl, heterocycloalkyl, cylcoalkenyl, heterocycloalkenyl,acyl, and aryl, any of which may be optionally substituted. In apreferred embodiment, R₁, if present, is aryl, substituted aryl,heteroaryl or alkyl; and R₂, if present is a hydrophobic group such asaryl, substituted aryl, heteroaryl or alkyl.

FIG. 13 shows the chemical structure of the compound HL2-006-1, ananalog of S3I-201.

FIG. 14 shows the chemical structure of the compound HL2-006-2, ananalog of S3I-201.

FIG. 15 shows the chemical structure of the compound HL2-006-3, ananalog of S3I-201.

FIG. 16 shows the chemical structure of the compound HL2-006-4, ananalog of S3I-201.

FIG. 17 shows the chemical structure of the compound HL2-006-5, ananalog of S3I-201.

FIG. 18 shows the chemical structure of the compound HL2-011-1, ananalog of S3I-201.

FIG. 19 shows the chemical structure of the compound HL2-011-2, ananalog of S3I-201.

FIG. 20 shows the chemical structure of the compound HL2-011-3, ananalog of S3I-201.

FIG. 21 shows the chemical structure of the compound HL2-001-4, ananalog of S3I-201.

FIG. 22 shows the chemical structure of the compound HL2-011-5, ananalog of S3I-201.

FIG. 23 shows the chemical structure of the compound BG2069-1, an analogof S3I-201.

FIG. 24 shows the chemical structure of the compound HL2-011-6, ananalog of S3I-201.

FIG. 25 shows the chemical structure of the compound HL2-011-7, ananalog of S3I-201.

FIG. 26 shows the chemical structure of the compound HL2-005, an analogof S3I-201.

FIG. 27 shows the chemical structure of the compound HL2-003, an analogof S3I-201.

FIG. 28 shows the chemical structure of the compound BG2066, an analogof S3I-201.

FIG. 29 shows the chemical structure of the compound BG2074, an analogof S3I-201.

FIG. 30 shows the chemical structure of the compound BG3004, an analogof S3I-201.

FIG. 31 shows the chemical structure of the compound BG3006A, an analogof S3I-201.

FIG. 32 shows the chemical structure of the compound BG3006B, an analogof S3I-201.

FIG. 33 shows the chemical structure of the compound BG3006D, an analogof S3I-201.

FIG. 34 shows the chemical structure of the compound BG3009, an analogof S3I-201.

FIG. 35 shows the chemical structure of compounds RPM381, RPM384, andRPM385, wherein X=H, CO₂Et, and CO₂H, respectively.

FIG. 36 shows the chemical structure of compounds RPM405 and RPM411,wherein X=CO₂Et and CO₂H, respectively.

FIG. 37 shows the chemical structure of compounds RPM407 and RPM412,wherein X=CO₂Et and CO₂H, respectively.

FIG. 38 shows the chemical structure of compounds RPM408 and RPM410,wherein X=CO₂Et and CO₂H, respectively.

FIG. 39 shows the chemical structure of compounds RPM415 and RPM416,wherein X=CO₂Et and CO₂H, respectively.

FIG. 40 shows the chemical structure of compounds RPM418 and RPM418-A,wherein X=CO₂Et and CO₂H, respectively.

FIG. 41 shows the chemical structure of compound RPM427, wherein X=CO₂H.

FIG. 42 shows the chemical structure of compound RPM431, whereinX=CO₂Me.

FIG. 43 shows the chemical structure of compound RPM432, wherein X=CO₂H.

FIG. 44 shows the chemical structure of compounds RPM444 and RPM448,wherein X=CO₂Et and CO₂H, respectively.

FIG. 45 shows the chemical structure of compounds RPM445 and RPM447,wherein X=CO₂Et and X=CO₂H, respectively.

FIG. 46 shows the chemical structure of compound RPM452, wherein X=CO₂H.

FIG. 47 shows the chemical structure of compounds RPM202.

FIG. 48 shows the chemical structure of NSC-263435.

FIG. 49 shows the chemical structure of NSC-11421.

FIG. 50 shows the chemical structure of NSC-75912.

FIG. 51 shows the chemical structure of NSC-91529.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is the native pTyr-peptide (APY*LKT; *=phosphorylation).

SEQ ID NO:2 is the biotinylated conjugate pTyr-peptide (EPQpYEEIEL(where pY represents pTyr)).

SEQ ID NO:3 is the sequence of the hSIE (high affinity sis-inducibleelement from the c-fos gene, m67 variant) oligonucleotide probe.

SEQ ID NO:4 is the sequence of the MGFe (mammary gland factor elementfrom the bovine β-casein gene promoter) oligonucleotide probe.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns isolated compounds, compositionscomprising these compounds, and methods of using these compounds andcompositions as inhibitors of Stat3 and inhibitors of aberrant cellgrowth, e.g., as anti-cancer agents.

Constitutively-active Stat3 is a prevalent molecular abnormality with acritical role in human malignant transformation, and which represents avalid target for novel anticancer drug design. S3I-201 (NSC 74859) is anovel inhibitor of Stat3 activity identified from the National CancerInstitute chemical libraries using structure-based virtual screeningwith a computer model of the Stat3 SH2 domain bound to its Stat3phosphotyrosine peptide derived from the X-ray crystal structure of theStat3(3 homodimer. S3I-201 inhibits Stat3:Stat3 complex formation, andStat3 DNA-binding and transcriptional activities. Furthermore, S3I-201inhibits growth and induces apoptosis preferentially in tumors cellsthat contain persistently activated Stat3. Constitutively-dimerized andactive Stat3C and Stat3 SH2 domain rescue tumor cells fromS3I-201-induced apoptosis. Finally, S3I-201 inhibits the expression ofthe Stat3-regulated genes Cyclin D1, Bcl-xL and Survivin, and inhibitsthe growth of human breast tumors in vivo. These findings stronglysuggest that the antitumor activity of S3I-201 is mediated in partthrough inhibition of aberrant Stat3 activation and provide theproof-of-concept for the potential clinical use of Stat3 inhibitors,such as S3I-201, in tumors harboring aberrant Stat3.

Aspects of the invention include, but are not limited to, Stat3inhibitors, compositions comprising these compounds, and methods ofusing these compounds and compositions as inhibitors of Stat3 and/or asinhibitors of aberrant cell growth, e.g., as anti-cancer agents. In oneembodiment, the compound has a structure encompassed by Formula A, B, C,D, E, or F in FIGS. 10-12A-D, respectively, or a pharmaceuticallyacceptable salt or analog thereof. In another embodiment, the compoundis NSC 74859 (S3I-201; shown in FIG. 7), NSC 59263 (shown in FIG. 8),NSC 42067 (shown in FIG. 9), NSC 75912 (shown in FIG. 50), NSC 11421(shown in FIG. 49), NSC 91529 (shown in FIG. 51), NSC 263435 (shown inFIG. 48), or a pharmaceutically acceptable salt or analog of any of theforegoing. In another embodiment, the compound is an analog of S3I-201shown in FIGS. 13-47, i.e., a compound selected from the groupconsisting of HL2-006-1, HL2-006-2, HL2-006-3, HL2-006-4, HL2-006-5,HL2-011-1, HL2-011-2, HL2-011-3, HL2-011-4, HL2-011-5, BG2069-1,HL2-011-6, HL2-011-7, HL2-005, HL2-003, BG2066, BG2074, BG3004, BG3006A,BG3006B, BG3006D, BG3009, RPM381, RPM384, RPM385, RPM405, RPM411,RPM407, RPM412, RPM408, RPM410, RPM415, RPM416, RPM418, RPM418-A,RPM427, RPM431, RPM432, RPM444, RPM448, RPM445, RPM447, RPM452, andRPM202, or a pharmaceutically acceptable salt or analog of any of theforegoing. In another embodiment, the compound is one listed in Table 4,or a pharmaceutically acceptable salt or analog thereof.

One aspect of the subject invention provides methods for using thecompounds of the invention as Stat3 inhibitors and/or asanti-proliferative agents. Thus, in one embodiment, the method of theinvention comprises administering a compound of the invention to cellsin vitro or in vivo in an amount sufficient to achieve the desiredresult, e.g., reduction of Stat3 activation. In another embodiment, themethod comprises administering a compound of the invention to a human ornon-human subject in an amount effective to achieve the desiredtherapeutic result. In one embodiment, more than one compound of theinvention is administered to the cells in vitro or in vivo. In apreferred embodiment, the compound is S3I-201, or a pharmaceuticallyacceptable salt or analog thereof.

As used herein, the terms “treatment” and “treating”, and grammaticalvariations thereof, include therapy and prophylaxis. When used as atherapy, the compounds of the invention, by themselves or in conjunctionwith other agents, alleviate or reduce one or more symptoms associatedwith a proliferation disorder (e.g., cancer). Thus, the treatmentmethods may or may not be curative in nature. When used as aprophylactic treatment, the compounds of the invention, by themselves orin conjunction with other agents, delay the onset of (and may prevent)one or more symptoms associated with a proliferation disorder (e.g.,cancer), or may prevent the genesis of the condition.

In one aspect, the method of the invention is a method for treating aproliferation disorder, such as cancer, comprising administering aneffective amount of a compound of the invention to a subject in needthereof.

In another aspect, the method of the invention is a method forinhibiting the growth of cancer cells in vitro or in vivo, comprisingadministering an effective amount of a compound of the invention to thecancer cells.

In another aspect, the subject invention provides compositionscomprising at least one isolated compound of the invention, and apharmaceutically acceptable carrier.

By inhibiting the growth of cells proliferating in an aberrant manner,the methods, compounds, and compositions of the present invention can beused to treat a number of cell proliferation disorders, such as cancers,including, but not limited to, leukemias and lymphomas, such as acutelymphocytic leukemia, acute non-lymphocytic leukemias, chroniclymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's Disease,non-Hodgkin's lymphomas, and multiple myeloma, childhood solid tumorssuch as brain tumors, neuroblastoma, retinoblastoma, Wilms' Tumor, bonetumors, and soft-tissue sarcomas, common solid tumors of adults such aslung cancer, colon and rectum cancer, breast cancer, prostate cancer,urinary cancers, uterine cancers, bladder cancers, oral cancers,pancreatic cancer, melanoma and other skin cancers, stomach cancer,ovarian cancer, brain tumors, liver cancer, laryngeal cancer, thyroidcancer, esophageal cancer, and testicular cancer. The methods of thesubject invention can be carried out in vivo or in vitro, to inhibit thegrowth of cells (e.g., cancer cells) in humans and non-human mammals.Treatment for a proliferation disorder can proceed by the Stat3inhibitor's anti-proliferative activity such as pro-apoptotic activity,or by other mechanisms. In one embodiment, the proliferation disorder isone on which the Stat3 inhibitor(s) act by inhibition of Stat3DNA-binding.

Compounds of the invention having the capability to modulate (e.g.,reduce or eliminate) signaling of the STAT3 and/or STAT5 signalingpathway in vitro and/or in vivo, or to inhibit the growth of cancercells in vitro and/or in vivo by inhibition of STAT3 and/or STAT5signaling or a different mechanism, would be considered to have thedesired biological activity in accordance with the subject invention.For therapeutic applications, compounds of the subject invention havethe capability to inhibit activation of the STAT3 and/or STAT5 signalingpathway, or to inhibit the growth of cancer cells in vitro and/or invivo by inhibition of STAT3 and/or STAT5 signaling or a differentmechanism. Inhibition of STAT3 and/or STAT5 signaling can be assesseddirectly or indirectly by various methods, including assays forinhibition of STAT3 dimerization, inhibition of STAT3 DNA-binding,and/or inhibition of STAT5 DNA-binding, for example. Inhibition of STAT3and/or STAT5 signaling by compounds of the invention selectivelypromotes apoptosis in tumor cells that harbor constitutively activatedSTAT3. Therefore, the desirable goals of promoting apoptosis(“programmed cell death”) of selective cancerous cells and suppressionof malignant transformation of normal cells within a patient arelikewise accomplished through administration of antagonists orinhibitors of STAT 3 signaling of the present invention, which can beadministered as simple compounds or in a pharmaceutical formulation.

In one embodiment, the proliferation disorder to be treated is a cancerproducing a tumor characterized by over-activation of Stat1, Stat3,Stat5, or a combination of two or all three of the foregoing. Examplesof such cancer types include, but are not limited to, breast cancer,ovarian cancer, multiple myeloma and blood malignancies, such as acutemyelogenous leukemia.

In addition to cancer, the proliferation disorder to be treated usingthe compounds, compositions, and methods of the invention can be onecharacterized by aberrant Stat3 activation within cells associated witha non-malignant disease, pathological state or disorder (collectively“disease”), and likewise comprising administering or contacting thecells with a an effective amount of one or more Stat3 inhibitors toreduce or inhibit the proliferation. The proliferation, hypertrophy orovergrowth of cells that is common to these diseases is mediated byoveractivation of Stat3. This protein becomes activated by a series ofbiochemical events. The activation of Stat3 then leads to another seriesof inter-related biochemical reactions or signal transduction cascadesthat ultimately produce cell growth and division.

In one embodiment, the proliferation disorder to be treated ischaracterized by a proliferation of T-cells such as autoimmune disease,e.g., type 1 diabetes, lupus and multiple sclerosis, and pathologicalstates such as graft rejection induced by the presentation of a foreignantigen such as a graft in response to a disease condition (e.g., kidneyfailure). Other non-malignant diseases characterized by proliferation ofcells include cirrhosis of the liver and restenosis.

The methods of the present invention can be advantageously combined withat least one additional treatment method, including but not limited to,chemotherapy, radiation therapy, or any other therapy known to those ofskill in the art for the treatment and management of proliferationdisorders such as cancer.

In one embodiment, the methods and compositions of the invention includethe incorporation of a ras antagonist. Ras protein is the on/off switchbetween hormone/growth factor receptors and the regulatory cascadingthat result in cell division. For Ras to be activated (i.e., turned on)to stimulate the regulatory cascades, it must first be attached to theinside of the cell membrane. Ras antagonist drug development aimed atblocking the action of Ras on the regulatory cascades has focused oninterrupting the association of Ras with the cell membrane, blockingactivation of Ras or inhibiting activated Ras. The details of theapproaches to development of Ras antagonists are reviewed in Kloog, etal., Exp. Opin. Invest. Drugs, 1999, 8(12):2121-2140. Thus, by the term“ras antagonist”, it is meant any compound or agent that targets one ormore of these phenomena so as to result in inhibition of cellproliferation.

The Ras antagonists that may be used in conjunction with the Stat3inhibitors of the invention affect (e.g., inhibit) the binding of Ras tothe cell membrane, which in turn reduces or inhibits the unwanted cellproliferation. Preferred Ras antagonists include farnesyl thiosalicylicacid (FTS) and structurally related compounds or analogs thereof, whichare believed to function by displacing or dislodging Ras from itsmembrane anchor. These organic compounds may be administeredparenterally or orally. In a particularly preferred embodiment, the Rasantagonist is formulated for oral or parenteral administration bycomplexation with cyclodextrin.

While compounds of the invention can be administered to cells in vitroand in vivo as isolated compounds, it is preferred to administer thesecompounds as part of a pharmaceutical composition. The subject inventionthus further provides compositions comprising a compound of theinvention, such as those shown in FIGS. 7-9 (NSC 74859 (S3I-201), NSC59263, NSC 42067), FIGS. 48-51 (NSC 42067, NSC 75912, NSC 11421 NSC91529, and NSC 263435), FIGS. 10-12A-D (Formulas A, B, C, D, E, and F),FIGS. 13-47 (HL2-006-1, HL2-006-2, HL2-006-3, HL2-006-4, HL2-006-5,HL2-011-1, HL2-011-2, HL2-011-3, HL2-011-4, HL2-011-5, BG2069-1,HL2-011-6, HL2-011-7, HL2-005, HL2-003, BG2066, BG2074, BG3004, BG3006A,BG3006B, BG3006D, BG3009, RPM381, RPM384, RPM385, RPM405, RPM411,RPM407, RPM412, RPM408, RPM410, RPM415, RPM416, RPM418, RPM418-A,RPM427, RPM431, RPM432, RPM444, RPM448, RPM445, RPM447, RPM452, andRPM202), and listed in Tables 4 and 5, or physiologically acceptablesalt(s) or analogs of any of the foregoing; in association with at leastone pharmaceutically acceptable carrier. The pharmaceutical compositioncan be adapted for various routes of administration, such as enteral,parenteral, intravenous, intramuscular, topical, subcutaneous, and soforth. Administration can be continuous or at distinct intervals, as canbe determined by a person of ordinary skill in the art.

The compounds of the invention can be formulated according to knownmethods for preparing pharmaceutically useful compositions. Formulationsare described in a number of sources which are well known and readilyavailable to those skilled in the art. For example, Remington'sPharmaceutical Science (Martin, E. W., 1995, Easton Pa., Mack PublishingCompany, 19^(th) ed.) describes formulations which can be used inconnection with the subject invention. Formulations suitable foradministration include, for example, aqueous sterile injectionsolutions, which may contain antioxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic with the blood of theintended recipient; and aqueous and nonaqueous sterile suspensions whichmay include suspending agents and thickening agents. The formulationsmay be presented in unit-dose or multi-dose containers, for examplesealed ampoules and vials, and may be stored in a freeze dried(lyophilized) condition requiring only the condition of the sterileliquid carrier, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powder, granules, tablets, etc. It should be understood that inaddition to the ingredients particularly mentioned above, thecompositions of the subject invention can include other agentsconventional in the art having regard to the type of formulation inquestion.

The compounds of the present invention include all hydrates and salts ofthe compounds shown in Figured 7-9 (NSC 74859 (S3I-201), NSC 59263, NSC42067), FIGS. 10-12A-D (Formulas A, B, C, D, E, and F), FIGS. 48-51 (NSC42067, NSC 75912, NSC 11421 NSC 91529, and NSC 263435), or FIGS. 13-47(e.g., HL2-006-1, HL2-006-2, HL2-006-3, HL2-006-4, HL2-006-5, HL2-011-1,HL2-011-2, HL2-011-3, HL2-011-4, HL2-011-5, BG2069-1, HL2-011-6,HL2-011-7, HL2-005, HL2-003, BG2066, BG2074, BG3004, BG3006A, BG3006B,BG3006D, BG3009, RPM381, RPM384, RPM385, RPM405, RPM411, RPM407, RPM412,RPM408, RPM410, RPM415, RPM416, RPM418, RPM418-A, RPM427, RPM431,RPM432, RPM444, RPM448, RPM445, RPM447, RPM452, and RPM202), and listedin Tables 4 and 5, or of their analogs, that can be prepared by those ofskill in the art. Under conditions where the compounds of the presentinvention are sufficiently basic or acidic to form stable nontoxic acidor base salts, administration of the compounds as salts may beappropriate. Examples of pharmaceutically acceptable salts are organicacid addition salts formed with acids that form a physiologicalacceptable anion, for example, tosylate, methanesulfonate, acetate,citrate, malonate, tartarate, succinate, benzoate, ascorbate,alpha-ketoglutarate, and alpha-glycerophosphate. Suitable inorganicsalts may also be formed, including hydrochloride, sulfate, nitrate,bicarbonate, and carbonate salts.

Pharmaceutically acceptable salts of compounds (e.g., Stat3 inhibitors)may be obtained using standard procedures well known in the art, forexample, by reacting a sufficiently basic compound such as an amine witha suitable acid affording a physiologically acceptable anion. Alkalimetal (for example, sodium, potassium or lithium) or alkaline earthmetal (for example calcium) salts of carboxylic acids can also be made.

As used herein, the term “analogs” refers to compounds which aresubstantially the same as another compound but which may have beenmodified by, for example, adding side groups, oxidation or reduction ofthe parent structure. Analogs of the Stat3 inhibitors shown in FIGS. 7-9(e.g., NSC 74859 (S3I-201), NSC 59263, and NSC 42067) FIGS. 49-52 (NSC42067, NSC 75912, NSC 11421 NSC 91529, and NSC 263435), and othercompounds disclosed herein can be readily prepared using commonly knownstandard reactions. These standard reactions include, but are notlimited to, hydrogenation, alkylation, acetylation, and acidificationreactions. Chemical modifications can be accomplished by those skilledin the art by protecting all functional groups present in the moleculeand deprotecting them after carrying out the desired reactions usingstandard procedures known in the scientific literature (Greene, T. W.and Wuts, P. G. M. “Protective Groups in Organic Synthesis” John Wiley &Sons, Inc. New York. 3rd Ed. pg. 819, 1999; Honda, T. et al. Bioorg.Med. Chem. Lett., 1997, 7:1623-1628; Honda, T. et al. Bioorg. Med. Chem.Lett., 1998, 8:2711-2714; Konoike, T. et al. J. Org. Chem., 1997,62:960-966; Honda, T. et al. J. Med. Chem., 2000, 43:4233-4246; each ofwhich are hereby incorporated herein by reference in their entirety).Analogs exhibiting the desired biological activity (such as induction ofapoptosis, cytotoxicity, cytostaticity, induction of cell cycle arrest,etc.) can be identified or confirmed using cellular assays or other invitro or in vivo assays. For example, assays that detect inhibition ofStat3 activation, G₂/M cell cycle arrest, and/or reduction of tumorgrowth may be utilized.

It will be appreciated that the compounds of the invention can containone or more asymmetrically substituted carbon atoms (i.e., carboncenters). The presence of one or more of the asymmetric centers in ananalog of the invention, can give rise to stereoisomers, and in eachcase, the invention is to be understood to extend to all suchstereoisomers, including enantiomers and diastereomers, and mixturesincluding racemic mixtures thereof.

FIGS. 12A-12D show formulas C—F, respectively, describing compounds ofthe invention. In FIGS. 12A-12D, the XNH groups have a transposedarrangement (NHX) with respect to one another.

Referring to FIGS. 12A-12D, R₁ and R₂, if present, can be an aliphaticor aromatic group; X, if present ═CO, SO₂, CONH, or alkyl; Z, ifpresent, is alkyl; and phosphate mimic=that shown in FIGS. 10 and 11,e.g., CO₂H, SO₃H, PO₃H, NO₂, CH₂CO₂H, CF₂CO₂H, or CF(CO₂H)₂ tetrazole.Preferably, R₂ is a hydrophobic group or part of a hydrophobic group. Inone embodiment, R₁, if present, is H, alkyl, alkenyl, cycloalkyl,heterocycloalkyl, cylcoalkenyl, heterocycloalkenyl, acyl, and aryl, anyof which may be optionally substituted; and R₂, if present is H, alkyl,alkenyl, cycloalkyl, heterocycloalkyl, cylcoalkenyl, heterocycloalkenyl,acyl, and aryl, any of which may be optionally substituted. In apreferred embodiment, R₁, if present, is aryl, substituted aryl,heteroaryl or alkyl; and R₂, if present is a hydrophobic group such asaryl, substituted aryl, heteroaryl or alkyl.

The term “alkyl group” is intended to mean a group of atoms derived froman alkane by the removal of one hydrogen atom. Thus, the term includesstraight or branched chain alkyl moieties including, for example,methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, pentyl, hexyl, andthe like. Preferred alkyl groups contain from 1 to about 14 carbon atoms(C₁₋₁₄ alkyl).

The term “aryl group” is intended to mean a group derived from anaromatic hydrocarbon by removal of a hydrogen from the aromatic system.Preferred aryl groups contain phenyl or substituted phenyl groups. Thus,the term “aryl” includes an aromatic carbocyclic radical having a singlering or two condensed rings. This term includes, for example, phenyl ornaphthyl.

The term “heteroaryl” refers to aromatic ring systems of five or moreatoms (e.g., five to ten atoms) of which at least one atom is selectedfrom O, N and S, and includes for example furanyl, thiophenyl, pyridyl,indolyl, quinolyl and the like.

The term “acyl group” is intended to mean a group having the formulaRCO—, wherein R is an alkyl group or an aryl group.

The term “alkenyl” refers to a straight or branched chain alkyl moietyhaving two or more carbon atoms (e.g., two to six carbon atoms, C₂₋₆alkenyl) and having in addition one double bond, of either E or Zstereochemistry where applicable. This term would include, for example,vinyl, 1-propenyl, 1- and 2-butenyl, 2-methyl-2-propenyl, etc.

The term “cycloalkyl” refers to a saturated alicyclic moiety havingthree or more carbon atoms (e.g., from three to six carbon atoms) andwhich may be optionally benzofused at any available position. This termincludes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,indanyl and tetrahydronaphthyl.

The term “heterocycloalkyl” refers to a saturated heterocyclic moietyhaving three or more carbon atoms (e.g., from three to six carbon atoms)and one or more heteroatom from the group N, O, S (or oxidized versionsthereof) and which may be optionally benzofused at any availableposition. This term includes, for example, azetidinyl, pyrrolidinyl,tetrahydrofuranyl, piperidinyl, indolinyl and tetrahydroquinolinyl.

The term “cycloalkenyl” refers to an alicyclic moiety having three ormore carbon atoms (e.g., from three to six carbon atoms) and having inaddition one double bond. This term includes, for example, cyclopentenylor cyclohexenyl.

The term “heterocycloalkenyl” refers to an alicyclic moiety having fromthree to six carbon atoms and one or more heteroatoms from the group N,O, S (or oxides thereof) and having in addition one double bond. Thisterm includes, for example, dihydropyranyl.

The term “halogen” means a halogen of the periodic table, such asfluorine, chlorine, bromine, or iodine.

The term “optionally substituted” means optionally substituted with oneor more of the aforementioned groups (e.g., alkyl, aryl, heteroaryl,acyl, alkenyl, cycloalkyl, heterocycloalkyl, cycloalkenyl,heterocycloalkenyl, or halogen), at any available position or positions.

Specifically, “alkyl” can include, for example, methyl, ethyl, propyl,isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, hexyl, heptyl,octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl orpentadecyl; “alkenyl” can include vinyl, 1-propenyl, 2-propenyl,1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl,4-pentenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl,1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 1-nonenyl,2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl,8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl,6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl; 1-undecenyl, 2-undecenyl,3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl,8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl,3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl,8-dodecenyl, 9-dodecenyl, 10-dodecenyl, 11-dodecenyl, 1-tridecenyl,2-tridecenyl, 3-tridecenyl, 4-tridecenyl, 5-tridecenyl, 6-tridecenyl,7-tridecenyl, 8-tridecenyl, 9-tridecenyl, 10-tridecenyl, 11-tridecenyl,12-tridecenyl, 1-tetradecenyl, 2-tetradecenyl, 3-tetradecenyl,4-tetradecenyl, 5-tetradecenyl, 6-tetradecenyl, 7-tetradecenyl,8-tetradecenyl, 9-tetradecenyl, 10-tetradecenyl, 11-tetradecenyl,12-tetradecenyl, 13-tetradeceny, 1-pentadecenyl, 2-pentadecenyl,3-pentadecenyl, 4-pentadecenyl, 5-pentadecenyl, 6-pentadecenyl,7-pentadecenyl, 8-pentadecenyl, 9-pentadecenyl, 10-pentadecenyl,11-pentadecenyl, 12-pentadecenyl, 13-pentadecenyl, or 14-pentadecenyl;“alkoxy” can include methoxy, ethoxy, propoxy, isopropoxy, butoxy,iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, hexoxy, heptyloxy, octyloxy,nonyloxy, decyloxy, undecyloxy, dodecyloxy, tridecyloxy, tetradecyloxy,or pentadecyloxy; “alkanoyl” can include acetyl, propanoyl, butanoyl,pentanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl,undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, or pentadecanoyl;“cycloalkyl” can include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, or cyclooctyl, for example; “aryl” can includephenyl, indenyl, 5,6,7,8-tetrahydronaphthyl, or naphthyl, for example;and “heteroaryl” can include furyl, imidazolyl, tetrazolyl, pyridyl, (orits N-oxide), thienyl, pyrimidinyl (or its N-oxide), indolyl, orquinolyl (or its N-oxide), for example.

The compounds of the invention are useful for various non-therapeuticand therapeutic purposes. The compounds (e.g., Stat3 inhibitors) may beused for reducing aberrant cell growth in animals and humans. Because ofsuch anti-proliferative properties of the compounds, they are useful inreducing unwanted cell growth in a wide variety of settings including invitro and in vivo. In addition to their use in treatment methods, theStat3 inhibitors of the invention are useful as agents for investigatingthe role of Stat3 in cellular metabolism, and controlling Stat3-mediatedmalignant or non-malignant cell growth in vitro or in vivo. They arealso useful as standards and for teaching demonstrations.

Therapeutic application of the compounds and compositions comprisingthem can be accomplished by any suitable therapeutic method andtechnique presently or prospectively known to those skilled in the art.Further, the compounds of the invention can be used as startingmaterials or intermediates for the preparation of other useful compoundsand compositions.

Compounds of the invention (e.g., Stat3 inhibitors) may be locallyadministered at one or more anatomical sites, such as sites of unwantedcell growth (such as a tumor site, e.g., injected or topically appliedto the tumor), optionally in combination with a pharmaceuticallyacceptable carrier such as an inert diluent. Compounds of the inventionmay be systemically administered, such as intravenously or orally,optionally in combination with a pharmaceutically acceptable carriersuch as an inert diluent, or an assimilable edible carrier for oraldelivery. They may be enclosed in hard or soft shell gelatin capsules,may be compressed into tablets, or may be incorporated directly with thefood of the patient's diet. For oral therapeutic administration, theactive compound may be combined with one or more excipients and used inthe form of ingestible tablets, buccal tablets, troches, capsules,elixirs, suspensions, syrups, wafers, aerosol sprays, and the like.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac, or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the Stat3 inhibitor maybe incorporated into sustained-release preparations and devices.

The active agent (compounds of the invention) may also be administeredintravenously or intraperitoneally by infusion or injection. Solutionsof the active agent can be prepared in water, optionally mixed with anontoxic surfactant. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, triacetin, and mixtures thereof and inoils. Under ordinary conditions of storage and use, these preparationscan contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the compounds of the invention which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. The ultimatedosage form should be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. Optionally, the prevention of the action of microorganismscan be brought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars, buffers or sodium chloride. Prolongedabsorption of the injectable compositions can be brought about by theinclusion of agents that delay absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the compoundsof the invention in the required amount in the appropriate solvent withvarious other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

For topical administration, the compounds may be applied in pure-form,i.e., when they are liquids. However, it will generally be desirable toadminister them topically to the skin as compositions, in combinationwith a dermatologically acceptable carrier, which may be a solid or aliquid.

The compounds of the subject invention can be applied topically to asubject's skin to reduce the size (and may include complete removal) ofmalignant or benign growths. The compounds of the invention can beapplied directly to the growth. Preferably, the compound is applied tothe growth in a formulation such as an ointment, cream, lotion,solution, tincture, or the like. Drug delivery systems for delivery ofpharmacological substances to dermal lesions can also be used, such asthat described in U.S. Pat. No. 5,167,649 (Zook).

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the Stat3 inhibitor can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers, for example.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user. Examples of useful dermatological compositionswhich can be used to deliver the Stat3 inhibitors to the skin aredisclosed in Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat.No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Woltzman(U.S. Pat. No. 4,820,508).

Useful dosages of the pharmaceutical compositions of the presentinvention can be determined by comparing their in vitro activity, and invivo activity in animal models. Methods for the extrapolation ofeffective dosages in mice, and other animals, to humans are known to theart; for example, see U.S. Pat. No. 4,938,949.

Accordingly, the present invention includes a pharmaceutical compositioncomprising a compound of the invention in combination with apharmaceutically acceptable carrier. Pharmaceutical compositions adaptedfor oral, topical or parenteral administration, comprising an amount ofa compound of the invention constitute a preferred embodiment of theinvention. The dose administered to a patient, particularly a human, inthe context of the present invention should be sufficient to achieve atherapeutic response in the patient over a reasonable time frame,without lethal toxicity, and preferably causing no more than anacceptable level of side effects or morbidity. One skilled in the artwill recognize that dosage will depend upon a variety of factorsincluding the condition (health) of the subject, the body weight of thesubject, kind of concurrent treatment, if any, frequency of treatment,therapeutic ratio, as well as the severity and stage of the pathologicalcondition.

Depending upon the disorder or disease condition to be treated, asuitable dose(s) may be that amount that will reduce proliferation orgrowth of the target cell(s). In the context of cancer, a suitabledose(s) is that which will result in a concentration of the active agent(the compound of the invention) in cancer tissue, such as a malignanttumor, which is known to achieve the desired response. The preferreddosage is the amount which results in maximum inhibition of cancer cellgrowth, without unmanageable side effects. Administration of a compoundof the invention can be continuous or at distinct intervals, as can bedetermined by a person of ordinary skill in the art.

To provide for the administration of such dosages for the desiredtherapeutic treatment, in some embodiments, pharmaceutical compositionsof the invention can comprise between about 0.1% and 45%, andespecially, 1 and 15%, by weight of the total of one or more of thecompounds of the invention based on the weight of the total compositionincluding carrier or diluents. Illustratively, dosage levels of theadministered active ingredients can be: intravenous, 0.01 to about 20mg/kg; intraperitoneal, 0.01 to about 100 mg/kg; subcutaneous, 0.01 toabout 100 mg/kg; intramuscular, 0.01 to about 100 mg/kg; orally 0.01 toabout 200 mg/kg, and preferably about 1 to 100 mg/kg; intranasalinstillation, 0.01 to about 20 mg/kg; and aerosol, 0.01 to about 20mg/kg of animal (body) weight.

Mammalian species which benefit from the disclosed methods include, butare not limited to, primates, such as apes, chimpanzees, orangutans,humans, monkeys; domesticated animals (e.g., pets) such as dogs, cats,guinea pigs, hamsters, Vietnamese pot-bellied pigs, rabbits, andferrets; domesticated farm animals such as cows, buffalo, bison, horses,donkey, swine, sheep, and goats; exotic animals typically found in zoos,such as bear, lions, tigers, panthers, elephants, hippopotamus,rhinoceros, giraffes, antelopes, sloth, gazelles, zebras, wildebeests,prairie dogs, koala bears, kangaroo, opossums, raccoons, pandas, hyena,seals, sea lions, elephant seals, otters, porpoises, dolphins, andwhales. Other species that may benefit from the disclosed methodsinclude fish, amphibians, avians, and reptiles. As used herein, theterms “patient” and “subject” are used interchangeably and are intendedto include such human and non-human species. Likewise, in vitro methodsof the present invention can be carried out on cells of such human andnon-human species.

Patients in need of treatment using the methods of the present inventioncan be identified using standard techniques known to those in themedical or veterinary professions, as appropriate.

As used herein, the terms “cancer” and “cancerous” refer to or describethe physiological condition in mammals that is typically characterizedby unregulated cell growth. The cancer may be multi-drug resistant (MDR)or drug-sensitive. Examples of cancer include but are not limited to,carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particularexamples of such cancers include breast cancer, prostate cancer, coloncancer, squamous cell cancer, small-cell lung cancer, non-small celllung cancer, gastrointestinal cancer, pancreatic cancer, cervicalcancer, ovarian cancer, peritoneal cancer, liver cancer, e.g., hepaticcarcinoma, bladder cancer, colorectal cancer, endometrial carcinoma,kidney cancer, and thyroid cancer.

Other non-limiting examples of cancers are basal cell carcinoma, biliarytract cancer; bone cancer; brain and CNS cancer; choriocarcinoma;connective tissue cancer; esophageal cancer; eye cancer; cancer of thehead and neck; gastric cancer; intra-epithelial neoplasm; larynx cancer;lymphoma including Hodgkin's and Non-Hodgkin's lymphoma; melanoma;myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth,and pharynx); retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer ofthe respiratory system; sarcoma; skin cancer; stomach cancer; testicularcancer; uterine cancer; cancer of the urinary system, as well as othercarcinomas and sarcomas. Examples of cancer types that may potentiallybe treated using the Stat3 inhibitors of the present invention are alsolisted in Table 1.

TABLE 1 Examples of Cancer Types Acute Lymphoblastic Leukemia, AdultHairy Cell Leukemia Acute Lymphoblastic Leukemia, Head and Neck CancerChildhood Hepatocellular (Liver) Cancer, Adult Acute Myeloid Leukemia,Adult (Primary) Acute Myeloid Leukemia, Childhood Hepatocellular (Liver)Cancer, Childhood Adrenocortical Carcinoma (Primary) AdrenocorticalCarcinoma, Childhood Hodgkin's Lymphoma, Adult AIDS-Related CancersHodgkin's Lymphoma, Childhood AIDS-Related Lymphoma Hodgkin's LymphomaDuring Pregnancy Anal Cancer Hypopharyngeal Cancer Astrocytoma,Childhood Cerebellar Hypothalamic and Visual Pathway Glioma,Astrocytoma, Childhood Cerebral Childhood Basal Cell CarcinomaIntraocular Melanoma Bile Duct Cancer, Extrahepatic Islet Cell Carcinoma(Endocrine Pancreas) Bladder Cancer Kaposi's Sarcoma Bladder Cancer,Childhood Kidney (Renal Cell) Cancer Bone Cancer, Osteosarcoma/MalignantKidney Cancer, Childhood Fibrous Histiocytoma Laryngeal Cancer BrainStem Glioma, Childhood Laryngeal Cancer, Childhood Brain Tumor, AdultLeukemia, Acute Lymphoblastic, Adult Brain Tumor, Brain Stem Glioma,Leukemia, Acute Lymphoblastic, Childhood Childhood Leukemia, AcuteMyeloid, Adult Brain Tumor, Cerebellar Astrocytoma, Leukemia, AcuteMyeloid, Childhood Childhood Leukemia, Chronic Lymphocytic Brain Tumor,Cerebral Leukemia, Chronic Myelogenous Astrocytoma/Malignant Glioma,Leukemia, Hairy Cell Childhood Lip and Oral Cavity Cancer Brain Tumor,Ependymoma, Childhood Liver Cancer, Adult (Primary) Brain Tumor,Medulloblastoma, Liver Cancer, Childhood (Primary) Childhood LungCancer, Non-Small Cell Brain Tumor, Supratentorial Primitive LungCancer, Small Cell Neuroectodermal Tumors, Childhood Lymphoma,AIDS-Related Brain Tumor, Visual Pathway and Lymphoma, Burkitt'sHypothalamic Glioma, Childhood Lymphoma, Cutaneous T-Cell, see MycosisBrain Tumor, Childhood Fungoides and Sézary Syndrome Breast CancerLymphoma, Hodgkin's, Adult Breast Cancer, Childhood Lymphoma, Hodgkin's,Childhood Breast Cancer, Male Lymphoma, Hodgkin's During PregnancyBronchial Adenomas/Carcinoids, Lymphoma, Non-Hodgkin's, Adult ChildhoodLymphoma, Non-Hodgkin's, Childhood Burkitt's Lymphoma Lymphoma,Non-Hodgkin's During Carcinoid Tumor, Childhood Pregnancy CarcinoidTumor, Gastrointestinal Lymphoma, Primary Central Nervous SystemCarcinoma of Unknown Primary Macroglobulinemia, Waldenström's CentralNervous System Lymphoma, Malignant Fibrous Histiocytoma of PrimaryBone/Osteosarcoma Cerebellar Astrocytoma, Childhood Medulloblastoma,Childhood Cerebral Astrocytoma/Malignant Melanoma Glioma, ChildhoodMelanoma, Intraocular (Eye) Cervical Cancer Merkel Cell CarcinomaChildhood Cancers Mesothelioma, Adult Malignant Chronic LymphocyticLeukemia Mesothelioma, Childhood Chronic Myelogenous Leukemia MetastaticSquamous Neck Cancer with Chronic Myeloproliferative Disorders OccultPrimary Colon Cancer Multiple Endocrine Neoplasia Syndrome, ColorectalCancer, Childhood Childhood Cutaneous T-Cell Lymphoma, see MultipleMyeloma/Plasma Cell Neoplasm Mycosis Fungoides and Sezary MycosisFungoides Syndrome Myelodysplastic Syndromes Endometrial CancerMyelodysplastic/Myeloproliferative Diseases Ependymoma, ChildhoodMyelogenous Leukemia, Chronic Esophageal Cancer Myeloid Leukemia, AdultAcute Esophageal Cancer, Childhood Myeloid Leukemia, Childhood AcuteEwing's Family of Tumors Myeloma, Multiple Extracranial Germ Cell Tumor,Myeloproliferative Disorders, Chronic Childhood Nasal Cavity andParanasal Sinus Cancer Extragonadal Germ Cell Tumor NasopharyngealCancer Extrahepatic Bile Duct Cancer Nasopharyngeal Cancer, ChildhoodEye Cancer, Intraocular Melanoma Neuroblastoma Eye Cancer,Retinoblastoma Non-Hodgkin's Lymphoma, Adult Gallbladder CancerNon-Hodgkin's Lymphoma, Childhood Gastric (Stomach) Cancer Non-Hodgkin'sLymphoma During Pregnancy Gastric (Stomach) Cancer, Childhood Non-SmallCell Lung Cancer Gastrointestinal Carcinoid Tumor Oral Cancer, ChildhoodGerm Cell Tumor, Extracranial, Oral Cavity Cancer, Lip and ChildhoodOropharyngeal Cancer Germ Cell Tumor, ExtragonadalOsteosarcoma/Malignant Fibrous Germ Cell Tumor, Ovarian Histiocytoma ofBone Gestational Trophoblastic Tumor Ovarian Cancer, Childhood Glioma,Adult Ovarian Epithelial Cancer Glioma, Childhood Brain Stem OvarianGerm Cell Tumor Glioma, Childhood Cerebral Ovarian Low MalignantPotential Tumor Astrocytoma Pancreatic Cancer Glioma, Childhood VisualPathway and Pancreatic Cancer, Childhood Hypothalamic Pancreatic Cancer,Islet Cell Skin Cancer (Melanoma) Paranasal Sinus and Nasal CavityCancer Skin Carcinoma, Merkel Cell Parathyroid Cancer Small Cell LungCancer Penile Cancer Small Intestine Cancer Pheochromocytoma Soft TissueSarcoma, Adult Pineoblastoma and Supratentorial Primitive Soft TissueSarcoma, Childhood Neuroectodermal Tumors, Childhood Squamous CellCarcinoma, see Skin Pituitary Tumor Cancer (non-Melanoma) Plasma CellNeoplasm/Multiple Myeloma Squamous Neck Cancer with OccultPleuropulmonary Blastoma Primary, Metastatic Pregnancy and Breast CancerStomach (Gastric) Cancer Pregnancy and Hodgkin's Lymphoma Stomach(Gastric) Cancer, Childhood Pregnancy and Non-Hodgkin's LymphomaSupratentorial Primitive Primary Central Nervous System LymphomaNeuroectodermal Tumors, Childhood Prostate Cancer T-Cell Lymphoma,Cutaneous, see Rectal Cancer Mycosis Fungoides and Sezary Renal Cell(Kidney) Cancer Syndrome Renal Cell (Kidney) Cancer, ChildhoodTesticular Cancer Renal Pelvis and Ureter, Transitional Cell Thymoma,Childhood Cancer Thymoma and Thymic Carcinoma Retinoblastoma ThyroidCancer Rhabdomyosarcoma, Childhood Thyroid Cancer, Childhood SalivaryGland Cancer Transitional Cell Cancer of the Renal Salivary GlandCancer, Childhood Pelvis and Ureter Sarcoma, Ewing's Family of TumorsTrophoblastic Tumor, Gestational Sarcoma, Kaposi's Unknown Primary Site,Carcinoma of, Sarcoma, Soft Tissue, Adult Adult Sarcoma, Soft Tissue,Childhood Unknown Primary Site, Cancer of, Sarcoma, Uterine ChildhoodSezary Syndrome Unusual Cancers of Childhood Skin Cancer (non-Melanoma)Ureter and Renal Pelvis, Transitional Skin Cancer, Childhood Cell CancerUrethral Cancer Uterine Cancer, Endometrial Uterine Sarcoma VaginalCancer Visual Pathway and Hypothalamic Glioma, Childhood Vulvar CancerWaldenstrom's Macroglobulinemia Wilms' Tumor

As used herein, the term “tumor” refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues. For example, a particular cancer may becharacterized by a solid mass tumor. The solid tumor mass, if present,may be a primary tumor mass. A primary tumor mass refers to a growth ofcancer cells in a tissue resulting from the transformation of a normalcell of that tissue. In most cases, the primary tumor mass is identifiedby the presence of a cyst, which can be found through visual orpalpation methods, or by irregularity in shape, texture or weight of thetissue. However, some primary tumors are not palpable and can bedetected only through medical imaging techniques such as X-rays (e.g.,mammography) or magnetic resonance imaging (MRI), or by needleaspirations. The use of these latter techniques is more common in earlydetection. Molecular and phenotypic analysis of cancer cells within atissue can usually be used to confirm if the cancer is endogenous to thetissue or if the lesion is due to metastasis from another site. Thetreatment methods of the invention can be utilized for early, middle, orlate stage disease, and acute or chronic disease. In some embodiments,the tumor is characterized as one exhibiting aberrant activation ofStat3.

According to the method of the subject invention, a compound of theinvention can be administered to a patient by itself, or co-administeredwith one or more other agents such as another compound of the invention,or a different agent or agents. Co-administration can be carried outsimultaneously (in the same or separate formulations) or consecutively.Furthermore, according to the method of the subject invention, compoundsof the invention can be administered to a patient as adjuvant therapy.For example, compounds can be administered to a patient in conjunctionwith chemotherapy.

Thus, the compounds of the invention, whether administered separately,or as a pharmaceutical composition, can include various other componentsas additives. Examples of acceptable components or adjuncts which can beemployed in relevant circumstances include antioxidants, free radicalscavenging agents, peptides, growth factors, antibiotics, bacteriostaticagents, immunosuppressives, anticoagulants, buffering agents,anti-inflammatory agents, anti-angiogenics, anti-pyretics, time-releasebinders, anesthetics, steroids, and corticosteroids. Such components canprovide additional therapeutic benefit, act to affect the therapeuticaction of the compounds of the invention, or act towards preventing anypotential side effects which may be posed as a result of administrationof the compounds. The Stat3 inhibitors of the subject invention can beconjugated to a therapeutic agent, as well.

Additional agents that can be co-administered to target cells in vitroor in vivo, such as in a patient, in the same or as a separateformulation, include those that modify a given biological response, suchas immunomodulators. For example, proteins such as tumor necrosis factor(TNF), interferon (such as alpha-interferon and beta-interferon), nervegrowth factor (NGF), platelet derived growth factor (PDGF), and tissueplasminogen activator can be administered. Biological responsemodifiers, such as lymphokines, interleukins (such as interleukin-1(IL-1), interleukin-2 (IL-2), and interleukin-6 (IL-6)), granulocytemacrophage colony stimulating factor (GM-CSF), granulocyte colonystimulating factor (G-CSF), or other growth factors can be administered.In one embodiment, the methods and compositions of the inventionincorporate one or more agents selected from the group consisting ofanti-cancer agents, cytotoxic agents, chemotherapeutic agents,anti-signaling agents, and anti-angiogenic agents.

EXEMPLIFIED EMBODIMENTS

1. An isolated compound having the structure of S3I-201 (shown in FIG.7), NSC-59263 (shown in FIG. 8), NSC-42067 (shown in FIG. 9), Formula A(shown in FIG. 10), Formula B (shown in FIG. 11), Formula C (shown inFIG. 12A), Formula D (shown in FIG. 12B), Formula E, shown in FIG. 12C,Formula F (shown in FIG. 12D), NSC 75912 (shown in FIG. 50), NSC 11421(shown in FIG. 49), NSC 91529 (shown in FIG. 51), NSC 263435 (shown inFIG. 48), HL2-006-1 (shown in FIG. 13), HL2-006-2 (shown in FIG. 14),HL2-006-3 (shown in FIG. 15), HL2-006-4 (shown in FIG. 16), HL2-006-5(shown in FIG. 17), HL2-011-1 (shown in FIG. 18), HL2-011-2 (shown inFIG. 19), HL2-011-3 (shown in FIG. 20), HL2-011-4 (shown in FIG. 21),HL2-011-5 (shown in FIG. 22), BG2069-1 (shown in FIG. 23), HL2-011-6(shown in FIG. 24), HL2-011-7 (shown in FIG. 25), HL2-005 (shown in FIG.26), HL2-003 (shown in FIG. 27), BG2066 (shown in FIG. 28), BG2074(shown in FIG. 29), BG3004 (shown in FIG. 30), BG3006A (shown in FIG.31), BG3006B (shown in FIG. 32), BG3006D (shown in FIG. 33), BG3009(shown in FIG. 34), RPM381 (shown in FIG. 35), RPM384 (shown in FIG.35), RPM385 (shown in FIG. 35), RPM405 (shown in FIG. 36), RPM411 (shownin FIG. 36), RPM407 (shown in FIG. 37), RPM412 (shown in FIG. 37),RPM408 (shown in FIG. 38), RPM410 (shown in FIG. 38), RPM415 (shown inFIG. 39), RPM416 (shown in FIG. 39), RPM418 (shown in FIG. 40), RPM418-A(shown in FIG. 40), RPM427 (shown in FIG. 41), RPM431 (shown in FIG.42), RPM432 (shown in FIG. 43), RPM444 (shown in FIG. 44) RPM448 (shownin FIG. 44), RPM445 (shown in FIG. 45), RPM447 (shown in FIG. 45),RPM452 (shown in FIG. 46), RPM202 (shown in FIG. 47), a compound listedin Table 4, a compound listed in Table 5, or a pharmaceuticallyacceptable salt or analog of any of the foregoing.

2. A pharmaceutical composition comprising at least one isolatedcompound selected from selected from embodiment 1; and apharmaceutically acceptable carrier.

3. The composition of embodiment 2, wherein the compound is S3I-201.

4. The composition of embodiment 2 or 3, further comprising anadditional anti-cancer agent.

5. The composition of any of embodiments 2-4, further comprising anagent selected from the group consisting of an antioxidant, free radicalscavenging agent, peptide, growth factor, antibiotic, bacteriostaticagent, immunosuppressive, anticoagulant, buffering agent,anti-inflammatory agent, anti-angiogenic agent, anti-pyretic,time-release binder, anesthetic, steroid, and corticosteroid.

6. The composition of any of embodiments 2-5, comprising more than oneof said compounds.

7. A method of treating a proliferation disorder in a subject,comprising administering an effective amount of at least one compound ofembodiment 1 to the subject.

8. The method of embodiment 7, wherein said administering comprisingadministering an effective amount of S3I-201 to the subject.

9. The method of embodiment 7 or 8, wherein the proliferation disorderis cancer.

10. The method of any of embodiments 7-9, wherein the compound isadministered locally at the site of a tumor.

11. The method of any of embodiments 7-10, wherein the proliferationdisorder is cancer, and wherein the subject is suffering from a tumorand the compound inhibits growth of the tumor.

12. The method of embodiment 7 or 8, wherein the proliferation disorderis a non-malignant disease characterized by aberrant Stat3 activation ofcells.

13. The method of any of embodiments 7-12, wherein the compound isadministered locally at the site of the proliferation disorder.

14. The method of any of embodiments 7-9, wherein the subject is notsuffering from the proliferation disorder, and wherein the compound isadministered to delay onset of the proliferation disorder.

15. The method of any of embodiments 7-14, wherein the route ofadministration is selected from the group consisting of intravenous,intramuscular, oral, and intra-nasal.

16. The method of any of embodiments 7-15, wherein the subject is human.

17. The method of any of embodiments 7-15, wherein the subject is anon-human mammal.

18. The method of any of embodiments 7-13 or 15-17, further comprisingidentifying the subject as one suffering from the proliferationdisorder.

19. The method of embodiment 8, wherein the subject is suffering from atumor and wherein said administering comprises administering S3I-201 atthe site of the tumor.

20. A method of suppressing the growth of, or inducing apoptosis in,malignant cells, the method comprising contacting the cells with aneffective amount of at least one compound of embodiment 1.

21. The method of embodiment 20, wherein said administering is carriedout in vitro.

22. The method of embodiment 20, wherein said administering is carriedout in vivo.

23. The method of any of embodiments 20-22, wherein the cells aremammalian cancer cells.

24. The method of any of embodiments 20-23, wherein the cells consistessentially of human breast cancer cells.

25. The method of any of embodiments 20-24, wherein said contactingcomprises contacting the cells with an effective amount of S3I-201.

26. A method of inhibiting constitutive activation of Stat3 in cells,comprising contacting the cells with an effective amount of at least onecompound of embodiment 1.

27. The method of embodiment 26, wherein said administering is carriedout in vitro.

28. The method of embodiment 26, wherein said administering is carriedout in vivo.

29. The method of embodiment 27 or 28, wherein said contacting comprisescontacting the cells with an effective amount of S3I-201.

30. The method of any of embodiments 26-29, wherein the cells are cancercells.

31. The method of any of embodiments 26-30, wherein the cells consistessentially of human breast cancer cells.

32. A method of preventing Stat3 dimerization in a mammalian cell, themethod comprising contacting the cell with an effective amount of atleast compound of embodiment 1.

33. The method of embodiment 32, wherein said contacting comprisescontacting the cell with an effective amount of S3I-201.

34. A method of disrupting Stat3-DNA binding or Stat5-DNA binding, themethod comprising contacting the Stat3 or Stat5 with an effective amountof at least one compound of embodiment 1.

35. The method of embodiment 34, wherein said contacting comprisescontacting the Stat3 or Stat5 with an effective amount of S3I-201.

36. An in-vitro screening test for presence of malignant cells in amammalian tissue, the test comprising:

-   -   obtaining a sample containing viable cells of said tissue;    -   culturing said sample under conditions promoting growth of the        viable cells contained therein;    -   treating the cultured sample with a compound; and    -   analyzing the treated sample by a method effective to determine        percent apoptosis of cells as an indicator of presence of        malignant cells in the sample, wherein the compound is at least        one compound of embodiment 1.

37. The screening test of embodiment 36, wherein said treatingcomprising contacting the cells with S3I-201.

38. A method of identifying inhibitors of constitutive Stat3 activation,Stat3-DNA binding, Stat5-DNA binding, and/or Stat3 dimerization, themethod comprising selecting a compound having a structure of Formula A,B, C, D, E, or F (shown in FIGS. 10-12A-D); and determining whether thecompound inhibits constitutive Stat3 activation, disrupts Stat3-DNAbinding, disrupts Stat5-DNA binding, prevents Stat3 dimerization, ordetermining two or more of the foregoing.

39. The method of embodiment 38, wherein the compound is not oneselected from embodiment 1.

40. A method of identifying anti-cancer agents, the method comprisingselecting a compound having a structure of Formulas A, B, C, D, E, or F(shown in FIGS. 10-12A-D); and determining whether the compound inhibitsthe growth of cancer cells in vitro or in vivo.

41. The method of embodiment 41, wherein the compound is not oneselected from embodiment 1.

Assays known in the art and/or disclosed herein may be used to evaluatecell apoptosis and/or inhibition of Stat signaling in carrying out thein vitro screening test and methods set forth in the above embodiments,such as assays for inhibition of dimerization (see, for example, Schust,J., Berg, T., Analytical Biochemistry, 2004, 330(1):114-118, which isincorporated by reference herein in its entirety); and assays forinhibition of DNA binding (see, for example, Turkson J. et al., J. Biol.Chem., 2001, 276(48):45443-45455, which is incorporated by referenceherein in its entirety). Likewise, such assays may be used to confirmthe desired biological activity possessed by analogs of the invention.

Definitions

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the development or spread ofcancer or other proliferation disorder. For purposes of this invention,beneficial or desired clinical results include, but are not limited to,alleviation of symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. For example, treatment with a compound of the inventionmay include reduction of undesirable cell proliferation, and/orinduction of apoptosis and cytotoxicity. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment. Those in need of treatment include those already with thecondition or disorder as well as those prone to have the condition ordisorder or those in which the condition or disorder is to be preventedor onset delayed. Optionally, the patient may be identified (e.g.,diagnosed) as one suffering from the disease or condition (e.g.,proliferation disorder) prior to administration of the Stat3 inhibitorof the invention.

As used herein, the term “(therapeutically) effective amount” refers toan amount of the compound of the invention or other agent (e.g., a drug)effective to treat a disease or disorder in a mammal. In the case ofcancer or other proliferation disorder, the therapeutically effectiveamount of the agent may reduce (i.e., slow to some extent and preferablystop) unwanted cellular proliferation; reduce the number of cancercells; reduce the tumor size; inhibit (i.e., slow to some extent andpreferably stop) cancer cell infiltration into peripheral organs;inhibit (i.e., slow to some extent and preferably stop) tumormetastasis; inhibit, to some extent, tumor growth; reduce Stat3signaling in the target cells (such as by inhibiting the binding of DNAand Stat3), and/or relieve, to some extent, one or more of the symptomsassociated with the cancer. To the extent the administered compoundprevents growth of and/or kills existing cancer cells, it may becytostatic and/or cytotoxic. For cancer therapy, efficacy can, forexample, be measured by assessing the time to disease progression (TTP)and/or determining the response rate (RR).

As used herein, the term “growth inhibitory amount” of the compound ofthe invention refers to an amount which inhibits growth or proliferationof a target cell, such as a tumor cell, either in vitro or in vivo,irrespective of the mechanism by which cell growth is inhibited (e.g.,by cytostatic properties, cytotoxic properties, etc.). In a preferredembodiment, the growth inhibitory amount inhibits (i.e., slows to someextent and preferably stops) proliferation or growth of the target cellin vivo or in cell culture by greater than about 20%, preferably greaterthan about 50%, most preferably greater than about 75% (e.g., from about75% to about 100%).

The terms “cell” and “cells” are used interchangeably herein and areintended to include either a single cell or a plurality of cells, invitro or in vivo, unless otherwise specified.

As used herein, the term “anti-cancer agent” refers to a substance ortreatment that inhibits the function of cancer cells, inhibits theirformation, and/or causes their destruction in vitro or in vivo. Examplesinclude, but are not limited to, cytotoxic agents (e.g., 5-fluorouracil,TAXOL), chemotherapeutic agents, and anti-signaling agents (e.g., thePI3K inhibitor LY). In some embodiments, the anti-cancer agent is a rasantagonist.

As used herein, the term “cytotoxic agent” refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells in vitro and/or in vivo. The term is intended to includeradioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², and radioactive isotopes of Lu), chemotherapeutic agents,toxins such as small molecule toxins or enzymatically active toxins ofbacterial, fungal, plant or animal origin, and antibodies, includingfragments and/or variants thereof.

As used herein, the term “chemotherapeutic agent” is a chemical compounduseful in the treatment of cancer, such as, for example, taxanes, e.g.,paclitaxel (TAXOL, BRISTOL-MYERS SQUIBB Oncology, Princeton, N.J.) anddoxetaxel (TAXOTERE, Rhone-Poulenc Rorer, Antony, France), chlorambucil,vincristine, vinblastine, anti-estrogens including for exampletamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andtoremifene (FARESTON, GTx, Memphis, Tenn.), and anti-androgens such asflutamide, nilutamide, bicalutamide, leuprolide, and goserelin, etc.Examples of chemotherapeutic agents that may be used in conjunction withthe compounds of the invention are listed in Table 2. In a preferredembodiment, the chemotherapeutic agent is one or more anthracyclines.Anthracyclines are a family of chemotherapy drugs that are alsoantibiotics. The anthracyclines act to prevent cell division bydisrupting the structure of the DNA and terminate its function by: (1)intercalating into the base pairs in the DNA minor grooves; and (2)causing free radical damage of the ribose in the DNA. The anthracyclinesare frequently used in leukemia therapy. Examples of anthracyclinesinclude daunorubicin (CERUBIDINE), doxorubicin (ADRIAMYCIN, RUBEX),epirubicin (ELLENCE, PHARMORUBICIN), and idarubicin (IDAMYCIN).

TABLE 2 Examples of Chemotherapeutic Agents 13-cis-Retinoic Acid Mylocel2-Amino-6- Letrozole Mercaptopurine Neosar 2-CdA Neulasta2-Chlorodeoxyadenosine Neumega 5-fluorouracil Neupogen 5-FU Nilandron6-TG Nilutamide 6-Thioguanine Nitrogen Mustard 6-Mercaptopurine Novaldex6-MP Novantrone Accutane Octreotide Actinomycin-D Octreotide acetateAdriamycin Oncospar Adrucil Oncovin Agrylin Ontak Ala-Cort OnxalAldesleukin Oprevelkin Alemtuzumab Orapred Alitretinoin OrasoneAlkaban-AQ Oxaliplatin Alkeran Paclitaxel All-transretinoic acidPamidronate Alpha interferon Panretin Altretamine ParaplatinAmethopterin Pediapred Amifostine PEG Interferon AminoglutethimidePegaspargase Anagrelide Pegfilgrastim Anandron PEG-INTRON AnastrozolePEG-L-asparaginase Arabinosylcytosine Phenylalanine Mustard Ara-CPlatinol Aranesp Platinol-AQ Aredia Prednisolone Arimidex PrednisoneAromasin Prelone Arsenic trioxide Procarbazine Asparaginase PROCRIT ATRAProleukin Avastin Prolifeprospan 20 with Carmustine implant BCGPurinethol BCNU Raloxifene Bevacizumab Rheumatrex Bexarotene RituxanBicalutamide Rituximab BiCNU Roveron-A (interferon alfa-2a) BlenoxaneRubex Bleomycin Rubidomycin hydrochloride Bortezomib SandostatinBusulfan Sandostatin LAR Busulfex Sargramostim C225 Solu-Cortef CalciumLeucovorin Solu-Medrol Campath STI-571 Camptosar StreptozocinCamptothecin-11 Tamoxifen Capecitabine Targretin Carac Taxol CarboplatinTaxotere Carmustine Temodar Carmustine wafer Temozolomide CasodexTeniposide CCNU TESPA CDDP Thalidomide CeeNU Thalomid CerubidineTheraCys cetuximab Thioguanine Chlorambucil Thioguanine TabloidCisplatin Thiophosphoamide Citrovorum Factor Thioplex CladribineThiotepa Cortisone TICE Cosmegen Toposar CPT-11 TopotecanCyclophosphamide Toremifene Cytadren Trastuzumab Cytarabine TretinoinCytarabine liposomal Trexall Cytosar-U Trisenox Cytoxan TSPA DacarbazineVCR Dactinomycin Velban Darbepoetin alfa Velcade Daunomycin VePesidDaunorubicin Vesanoid Daunorubicin Viadur hydrochloride VinblastineDaunorubicin liposomal Vinblastine Sulfate DaunoXome Vincasar PfsDecadron Vincristine Delta-Cortef Vinorelbine Deltasone Vinorelbinetartrate Denileukin diftitox VLB DepoCyt VP-16 Dexamethasone VumonDexamethasone acetate Xeloda dexamethasone sodium Zanosar phosphateZevalin Dexasone Zinecard Dexrazoxane Zoladex DHAD Zoledronic acid DICZometa Diodex Gliadel wafer Docetaxel Glivec Doxil GM-CSF DoxorubicinGoserelin Doxorubicin liposomal granulocyte-colony stimulating factorDroxia Granulocyte macrophage colony stimulating DTIC factor DTIC-DomeHalotestin Duralone Herceptin Efudex Hexadrol Eligard Hexalen EllenceHexamethylmelamine Eloxatin HMM Elspar Hycamtin Emcyt Hydrea EpirubicinHydrocort Acetate Epoetin alfa Hydrocortisone Erbitux Hydrocortisonesodium phosphate Erwinia L-asparaginase Hydrocortisone sodium succinateEstramustine Hydrocortone phosphate Ethyol Hydroxyurea EtopophosIbritumomab Etoposide Ibritumomab Tiuxetan Etoposide phosphate IdamycinEulexin Idarubicin Evista Ifex Exemestane IFN-alpha Fareston IfosfamideFaslodex IL-2 Femara IL-11 Filgrastim Imatinib mesylate FloxuridineImidazole Carboxamide Fludara Interferon alfa Fludarabine InterferonAlfa-2b (PEG conjugate) Fluoroplex Interleukin-2 FluorouracilInterleukin-11 Fluorouracil (cream) Intron A (interferon alfa-2b)Fluoxymesterone Leucovorin Flutamide Leukeran Folinic Acid Leukine FUDRLeuprolide Fulvestrant Leurocristine G-CSF Leustatin Gefitinib LiposomalAra-C Gemcitabine Liquid Pred Gemtuzumab ozogamicin Lomustine GemzarL-PAM Gleevec L-Sarcolysin Lupron Meticorten Lupron Depot MitomycinMatulane Mitomycin-C Maxidex Mitoxantrone Mechlorethamine M-PrednisolMechlorethamine MTC Hydrochlorine MTX Medralone Mustargen Medrol MustineMegace Mutamycin Megestrol Myleran Megestrol Acetate Iressa MelphalanIrinotecan Mercaptopurine Isotretinoin Mesna Kidrolase Mesnex LanacortMethotrexate L-asparaginase Methotrexate Sodium LCR Methylprednisolone

As used herein, the term “Stat” refers to signal transducers andactivators of transcription, which represent a family of proteins that,when activated by protein tyrosine kinases in the cytoplasm of the cell,migrate to the nucleus and activate gene transcription. Examples ofmammalian STATs include STAT 1, STAT2, STAT3, STAT4, STAT5a, STAT5b, andSTAT6.

As used herein, the term “signaling” and “signaling transduction”represents the biochemical process involving transmission ofextracellular stimuli, via cell surface receptors through a specific andsequential series of molecules, to genes in the nucleus resulting inspecific cellular responses to the stimuli.

As used herein, the term “constitutive activation,” as in theconstitutive activation of the STAT pathway, refers to a condition wherethere is an abnormally elevated level of tyrosine phosphorylated STAT3within a given cell(s), e.g., cancer cells, as compared to acorresponding normal (e.g., non-cancer or non-transformed) cell.Constitutive activation of STAT3 has been exhibited in a large varietyof malignancies, including, for example, breast carcinoma cell lines;primary breast tumor specimens; ovarian cancer cell lines and tumors;multiple myeloma tumor specimens; and blood malignancies, such as acutemyelogenous leukemia, as described in published PCT internationalapplication WO 00/44774 (Jove, R. et al.), the disclosure of which isincorporated herein by reference in its entirety.

Methods for determining whether a human or non-human mammalian subjecthas abnormally high levels of constitutively-activated Stat3 are knownin the art and are described, for example, in U.S. patent publication2004-0138189-A1 and PCT publication 02/078617 A, each of which areincorporated herein by reference in their entirety. Optionally, themethods of the invention further comprise identifying a patientsuffering from a condition (e.g., cancer) associated with an abnormallyelevated level of tyrosine phosphorylated STAT3, or determining whetherthe cancer cells can be characterized as having abnormally elevatedlevels of tyrosine phosphorylated Stat3.

As used herein, the term “pharmaceutically acceptable salt or prodrug”is intended to describe any pharmaceutically acceptable form (such as anester, phosphate ester, salt of an ester or a related group) of acompound of the invention, which, upon administration to a subject,provides the mature or base compound (e.g., a Stat3-inhibitorycompound). Pharmaceutically acceptable salts include those derived frompharmaceutically acceptable inorganic or organic bases and acids.Suitable salts include those derived from alkali metals such aspotassium and sodium, alkaline earth metals such as calcium andmagnesium, among numerous other acids well known in the pharmaceuticalart. Pharmaceutically acceptable prodrugs refer to a compound that ismetabolized, for example hydrolyzed or oxidized, in the host to form thecompound of the present invention. Typical examples of prodrugs includecompounds that have biologically labile protecting groups on afunctional moiety of the active compound. Prodrugs include compoundsthat can be oxidized, reduced, aminated, deaminated, hydroxylated,dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated,acylated, deacylated, phosphorylated, dephosphorylated to produce theactive compound.

The term “pharmaceutically acceptable esters” as used herein, unlessotherwise specified, includes those esters of one or more compounds,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of hosts without undue toxicity, irritation,allergic response and the like, are commensurate with a reasonablebenefit/risk ratio, and are effective for their intended use.

The terms “comprising”, “consisting of” and “consisting essentially of”are defined according to their standard meaning. The terms may besubstituted for one another throughout the instant application in orderto attach the specific meaning associated with each term.

The terms “isolated” or “biologically pure” refer to material that issubstantially or essentially free from components which normallyaccompany the material as it is found in its native state.

As used in this specification, the singular forms “a”, “an”, and “the”include plural reference unless the context clearly dictates otherwise.Thus, for example, a reference to “a compound” includes more than onesuch compound. A reference to “a Stat3 inhibitor” includes more than onesuch inhibitor, and so forth.

The practice of the present invention can employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA technology, electrophysiology, and pharmacology that arewithin the skill of the art. Such techniques are explained fully in theliterature (see, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning:A Laboratory Manual, Second Edition (1989); DNA Cloning, Vols. I and II(D. N. Glover Ed. 1985); Perbal, B., A Practical Guide to MolecularCloning (1984); the series, Methods In Enzymology (S. Colowick and N.Kaplan Eds., Academic Press, Inc.); Transcription and Translation (Hameset al. Eds. 1984); Gene Transfer Vectors For Mammalian Cells (J. H.Miller et al. Eds. (1987) Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.); Scopes, Protein Purification: Principles and Practice(2nd ed., Springer-Verlag); and PCR: A Practical Approach (McPherson etal. Eds. (1991) IRL Press)), each of which are incorporated herein byreference in their entirety.

Following are examples that illustrate materials, methods, andprocedures for practicing the invention. The examples are illustrativeand should not be construed as limiting.

Materials and Methods

Cells and Reagents.

Normal mouse fibroblasts (NIH3T3) and their counterparts transformed byv-Src (NIH3T3/v-Src), v-Ras (NIH3T3/v-Ras) or overexpressing the humanEGF receptor (NIH3T3/hEGFR), and the human breast cancer (MDA-MB-231,MDA-MB-435, MDA-MB-453, and MDA-MB-468) cells have all been previouslyreported (Turkson, J. et al. J. Biol. Chem., 2001, 276:45443-45455; Yu,C. L. et al. Science, 1995, 269:81-83; Garcia, R. et al. Oncogene, 2001,20:2499-2513; Johnson, P. J. et al. Mol. Cell. Biol., 1985,5:1073-1083). Cells were grown in Dulbecco's modified Eagle's medium(DMEM) containing 10% heat-inactivated fetal bovine serum. AntibodiesC-136 and E23× for Stat1, C20 and C20× for Stat3 and L-20 and L-20× forStat5A were obtained from Santa Cruz Biotechnology, Santa Cruz, Calif.,and polyclonal anti-Src and anti-phosphoSrc antibodies from CellSignaling Technology, Danvers, Mass.

Transient Transfection of Cells and Treatment with Compound.

Twelve to twenty-four hours following seeding, normal mouse fibroblasts(NIH3T3) were transiently co-transfected with 4 μg each of pLucTKS3,pLucSRE, or β-Casein Luc and 4 μg pMvSrc and 100 ng β-galactosidase (fornormalizing), or the human breast carcinoma MDA-MB-231 cells weretransiently transfected with pRc/CMV-Stat3C (kindly provided by J.Bromberg and J. Darnell (Bromberg, J. F. et al. Cell, 1999,98:295-303)), or with FLAG-tagged N-terminus of Stat3 (Stat3-NT) orFLAG-tagged Stat3 SH2 domain (ST3-SH2) (Zhang, T. et al. J. Biol. Chem.,2002, 277:17556-175563), or mock transfected for 4 hours usingLipofectamine plus (Invitrogen, Carlsbad, Calif.) and following themanufacturer's protocol. Twenty-four hours after transfection, cellswere untreated (0.05% DMSO) or treated with S3I-201 (100 μM) for anadditional 24 hours, harvested and cytosolic extracts prepared forluciferase assay, as previously done (Turkson, J. et al. J. Biol. Chem.,2001, 276:45443-45455; Turkson, J. et al. Mol. Cell. Biol., 1998,18:2545-2552) or cells were analyzed by Annexin V binding and FlowCytometry.

Cytosolic Extracts and Cell Lysates Preparation, Luciferase andβ-Galactosidase Assay.

Cytosolic extract preparation from mammalian cells for luciferase andβ-gal assays, and from recombinant baculovirus-infected Sf9 cells toobtain Stat1, Stat3, and Stat5 monomers, and Src proteins are asdescribed previously (Turkson, J. et al. J. Biol. Chem., 2001,276:45443-45455; Turkson, J. et al. Mol. Cell. Biol., 1998,18:2545-2552; Zhang, Y. et al. J. Biol. Chem., 2000, 275:24935-24944).Luciferase assays were done according to the supplier's (Promega,Madison, Wis.) manual and measured with a luminometer. β-gal assays wereperformed on lysates from transfected cells, as previously reported(Turkson, J. et al. J. Biol. Chem., 2001, 276:45443-45455; Turkson, J.et al. Mol. Cell. Biol., 1998, 18:2545-2552).

Nuclear Extract Preparation, Gel Shift Assays, and DensitometricAnalysis.

Nuclear extract preparations and electrophoretic mobility shift assay(EMSA) were carried out as previously described (Yu, C. L. et al.Science, 1995, 269:81-83; Garcia, R. et al. Oncogene, 2001,20:2499-2513; Turkson, J. et al. Mol. Cell. Biol., 1998, 18:2545-2552).The ³²P-labeled oligonucleotide probes used were hSIE (high affinitysis-inducible element from the c-fos gene, m67 variant,5′-AGCTTCATTTCCCGTAAATCCCTA) (SEQ ID NO:3) that binds Stat1 and Stat3(Garcia, R. et al. Oncogene, 2001, 20:2499-2513; Wagner, M. et al.Pancreas, 1999, 19:370-376) and MGFe (mammary gland factor element fromthe bovine β-casein gene promoter, 5′-AGATTTCTAGGAATTCAA) (SEQ ID NO:4)for Stat1 and Stat5 binding (Gouilleux, F. et al. Endocrinology, 1995,136:5700-5708; Seidel, H. M. et al. Proc. Natl. Acad. Sci. USA, 1995,92:3041-3045). Except where indicated, nuclear extracts werepre-incubated with compound for 30 minutes at room temperature prior toincubation with the radiolabeled probe. Bands corresponding toDNA-binding activities were scanned and quantified for eachconcentration of compound and plotted as percent of control (vehicle)against concentration of compound, from which the IC₅₀ values werederived, as previously reported (Turkson, J. et al. J. Biol. Chem.,2001, 276:45443-45455; Turkson, J. et al. J Biol Chem., 2005,280:32979-32988).

Immunoprecipitation (IP) and Western Blotting.

Whole-cell lysates and tumor tissue lysates from pulverized tumor tissuewere prepared in boiling SDS sample loading buffer to extract totalproteins, as previously described (Turkson, J. et al. Mol Cancer Ther,2004, 3:261-269; Turkson, J. et al. Mol. Cancer Ther., 2004,3:1533-1542; Turkson, J. et al. J Biol Chem., 2005, 280:32979-32988;Garcia, R. et al. Oncogene, 2001, 20:2499-2513). For IP, lysates wereprepared from NIH3T3/v-Src mouse fibroblasts overexpressing theFLAG-tagged Stat3 (FLAG-ST3) and Stat3-YFP in the IP buffer (25 mMTris-HCl, pH 7.2, 150 mM NaCl, 25 mM NaF, 0.5 mM Na orthovanadate, 20 mMPNPP, 1 mM benzamidine, 1 mM DTT, 1% Triton X-100, 2 μg/ml aprotinin, 2μg/ml leupeptin, 1 μg/ml pepstatin, 100 μg/ml PMSF). FLAG-ST3 andStat3-YFP were then immunoprecipitated by adding monoclonal anti-FLAG M2(Sigma-Aldrich, St. Louis, Mo.) or anti-YFP (Santa Cruz) antibody andincubating at 4° C. overnight with gentle rocking. Protein A/GPLUS-Agarose (Santa Cruz) was added at 1:10 and incubated with rockingfor 3 hours at 4° C. and centrifuged at 14,000 rpm at 4° C. for 30seconds. Bead pellets were washed 5× in the IP buffer, equivalent volumeof 3×SDS sample loading buffer added, vortexed, and boiled for 5minutes. Equivalent amounts of total protein were electrophoresed on anSDS-7.5-15% polyacrylamide gel and transferred onto nitrocellulosemembranes. Probing of nitrocellulose membranes with primary antibodiesand detection of horseradish peroxidase-conjugated secondary antibodiesby enhanced chemiluminescence (Amersham, Piscataway, N.J.) wereperformed, as described previously (Turkson, J. et al. Mol Cancer Ther,2004, 3:261-269; Turkson, J. et al. J Biol Chem., 2005, 280:32979-32988;Garcia, R. et al. Oncogene, 2001, 20:2499-2513). The probes used wereanti-cyclin D1, anti-Bcl-xL, anti-phosphoTyr-Stat3, anti-phospho-Shc,anti-Survivin, anti-Erk1/2, anti-phosphoTyr, 4G10 clone, andanti-β-actin (Cell Signaling Technology, Beverly, Mass.), anti-Stat3,anti-Stat5A, and anti-pErk1/2 (Santa Cruz), anti-Src (Cell SignalingTechnology), anti-FLAG M2 (Sigma), and anti-YFP (Santa Cruz).

Lck-SH2 Domain-Phosphopeptide Binding Assay.

In vitro ELISA study involving the Lck-SH2-GST protein and the conjugatepTyr peptide, biotinyl-ε-Ac-EPQpYEEIEL-OH (SEQ ID NO:2) (BachemBioscience, PA) was performed as previously described (Lee, T. R. andLawrence, D. S. J Med Chem., 2000, 43:1173-1179). Briefly, 100 μl ofbiotinyl-ε-Ac-EPQpYEEIEL-OH (Bachem) (in 50 mM Tris, 150 mM NaCl, pH7.5) was added to each well of a streptavidin-coated 96-well microtiterplates (Pierce, Rockford, Ill.) and incubated with shaking at 4° C.overnight. Then plates were rinsed with PBS-Tween 20 and then two timeswith 200 μl of BSA-T-PBS (0.2% BSA, 0.1% Tween 20, PBS). Then 50 μl ofLck-SH2-GST (Santa Cruz Biotechnology) fusion protein (6.4 ng/ml inBSA-T-PBS) was added to each well of the 96-well plate in the presenceand absence of 50 μl of S3I-201 (for 30 and 100 μM final concentrations)and the plate was shaken at room temperature for 4 hours. Aftersolutions were removed, each well was rinsed four times with BSA-T-PBS(200 μl) and 100 μl polyclonal rabbit anti-GST antibody (CHEMICON,Temecula, Calif.) (100 ng/ml in BSA-T-PBS) was added to each well,incubated at 4° C. overnight. Following washing with BSA-T-PBS, 100 μlof 200 ng/ml BSA-T-PBS horseradish peroxidase conjugated mouseanti-rabbit antibody (Amersham Biosciences) was added to each well andincubated for 45 minutes at room temperature. After 4 washing steps eachwith BSA-T-PBS and 3 washing steps each with PBS-T, 100 μl of peroxidasesubstrate (1-Step Turbo TMB-ELISA, Pierce) was added to each well andincubated for 5-15 minutes. Peroxidase reaction was stopped by adding100 μl 1M sulfuric acid solution and absorbance was read at 450 nm withan ELISA plate reader.

Soft-Agar Colony Formation Assay.

Colony formation assays were carried out in 6-well dishes, as describedpreviously (Turkson, J. et al. J. Biol. Chem., 2001, 276:45443-45455).Briefly, each well contained 1.5 ml of 1% agarose in Dulbeco's modifiedEagle's medium as the bottom layer and 1.5 ml of 0.5% agarose inDulbeco's modified Eagle's medium containing 4000 or 6000 NIH3T3/v-Srcor NIH3T3/v-Ras fibroblasts, respectively, as the top layer. Treatmentwith S3I-201 was initiated 1 day after seeding cells by adding 100 μl ofmedium with or without S3I-201, and repeating every 3 days, until largecolonies were evident. Colonies were quantified by staining with 20 μlof 1 mg/ml iodonitrotetrazolium violet, incubating at 37° C. overnight,and counting the next day.

Measurement of Apoptosis by Flow Cytometry.

Proliferating cells were treated with or without S3I-201 for up to 48hours. In some cases, cells were first transfected with Stat3C, ST3-NT,or ST3-SH2 domain or mock-transfected for 24 hours prior to treatmentwith compound for an additional 24-48 hours. Cells were then detachedand analyzed by Annexin V binding (BD Biosciences, San Diego) accordingto the manufacturer's protocol and Flow Cytometry to quantify thepercent apoptosis.

Mice and In Vivo Tumor Studies.

Six-week-old female athymic nude mice were purchased from Harlan(Indianapolis, Ind.) and maintained in the institutional animalfacilities approved by the American Association for Accreditation ofLaboratory Animal Care. Athymic nude mice were injected in the leftflank area s.c. with 5×10⁶ human breast cancer MDA-MB-231 cells in 100μL of PBS. After 5 to 10 days, tumors with a diameter of 3 mm wereestablished. Animals were given S3I-201 i.v. at 5 mg/kg every 2 or 3days for two weeks and monitored every 2 or 3 days. Animals werestratified so that the mean tumor sizes in all treatment were nearlyidentical. Tumor volume was calculated according to the formulaV=0.52×a²×b, where a, smallest superficial diameter, b, largestsuperficial diameter.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication.

Example 1—Computational Modeling and Virtual Screening

The computational modeling and virtual screening study used the GLIDE(Grid-based Ligand Docking from Energetics) software (Friesner, R. etal. J. Med. Chem., 2004, 47:1739-1749; Halgren, T. et al. J. Med. Chem.,2004, 47:1750-1759) (available from Schrödinger, L.L.C.) for the dockingsimulations, and relied on the X-ray crystal structure of the Stat3(3homodimer bound to DNA (Becker, S. et al. Nature, 1998, 394:145-151)determined at 2.25 Å resolution (1BG1 in the Protein Databank). Themodeling approach was used as a platform for structure-based virtualhigh throughput screening of the chemical libraries of the NCI DiversitySet (≈2,400 3D structures) and the NCI Plated Set (≈151,000 3Dstructures). For the virtual screening, DNA was removed and only one ofthe two monomers was employed (see FIGS. 1A and 1B). To validate thedocking approach, the native pTyr peptide, APY*LKT (SEQ ID NO: 1), wasextracted from the crystal structure of one of the monomers and dockedto the other monomer, whereby GLIDE produced a docking mode that closelyresembled the X-ray crystal structure (data not shown).Three-dimensional structures of compounds from the NCI's chemicallibraries were downloaded from the NCI DTP website and processed withLigPrep (available from Schrödinger, L.L.C.) to produce 2,392 3Dstructures for the Diversity Set and 150,829 3D structures for thePlated Set. Then GLIDE 2.7 SP (Standard Precision mode) docked eachchemical structure (for small-molecule) into the pTyr peptide bindingsite within the SH2 domain of the monomer in order to obtain the bestdocking mode and docking score.

For stronger interactions of small-molecules within the Stat3 SH2 domainrelative to the native pTyr peptide, emphasis is placed on maintainingthe critical atomic contacts that make the greatest contribution to theoverall binding free energy. Strong hydrogen bonding and hydrophobicinteractions within the SH2 domain are observed for compounds identifiedwith good docking scores, which will be predicted to be strong bindersto Stat3 and potent Stat3 inhibitors. The best score observed from thedocking studies was −11.7 kcal/mol, relative to the nativephosphopeptide sequence APY*LKT (SEQ ID NO:1) (scored as −11.9kcal/mol). Typically, compounds receiving highly favorable scores (morenegative values) have structural features that include sulfonyl,carboxyl and hydroxyl functional groups. For example, the current hit,S3I-201 (NSC 74859), contains all 3 groups (FIG. 7), and from themodeling data the carboxylate moiety of S3I-201 is predicted to interactwith Ser613, Ser611, and Arg609, whereas the phenolic hydroxyl groupinteracts with Lys591 of the phosphotyrosine binding site of the Stat3SH2 domain.

Example 2—Identification of a Novel Chemical Probe as an Inhibitor ofStat3 DNA-Binding Activity

The best scoring compounds from the virtual screening studies wereselected for experimental analysis using an in vitro Stat3 DNA-bindingassay. Nuclear extracts containing activated STATs were incubated for 30minutes with or without increasing concentrations of compounds prior toincubation with the radiolabeled hSIE probe that binds to Stat1 and toStat3 or the MGFe probe that binds to Stat1 and to Stat5 and subjectedto EMSA analysis, as described under “Materials and Methods”. Resultsfor the confirmed hit, S3I-201 (FIG. 7), show differential inhibition ofDNA-binding activities of STATs. FIG. 2A, left panel shows potentinhibition of Stat3 DNA-binding activity by S3I-201 with an average IC₅₀value of 86+33 μM (Table 3). For selectivity against STAT familymembers, nuclear extract preparations from EGF-stimulated mousefibroblasts over-expressing the human epidermal growth factor receptor(NIH3T3/hEGFR) containing activated Stat1, Stat3, and Stat5 werepre-incubated with or without S3I-201 prior to incubation with theradiolabeled probes, as described in “Materials and Methods”. EMSAanalysis of the DNA-binding activities shows Stat3:Stat3 (upper),Stat1:Stat3 (intermediate) and Stat1:Stat1 (lower) bands of complexeswith the hSIE probe (FIG. 2A-2) and Stat5:Stat5 (upper) and Stat1:Stat1(lower) bands of complexes with the MGFe probe (FIG. 2A-2). S3I-201preferentially inhibits Stat3 DNA-binding activity over that of Stat1,and inhibits that of Stat5 with a 2-fold less potency (FIG. 2A-2 andTable 3). The appearance of different degrees of activity of S3I-201 at300 μM is due to the fact that different nuclear extract preparationswere used, one from the v-Src transformed mouse fibroblasts(NIH3T3/v-Src) containing only activated Stat3 (FIG. 2A-1) and the otherfrom the EGF-stimulated NIHT3T3/hEGFR that contains activated Stat1,Stat3, and Stat5 (FIG. 2A-2). Supershift analysis with anti-Stat3antibody shows protein:hSIE complex (FIG. 2A-1) contains Stat3, whileuse of anti-Stat1 antibody or anti-Stat5 antibody confirms protein:MGFecomplexes contain Stat1 or Stat5, respectively (FIG. 2A-2). Thesestudies have identified S3I-201 from the NCI chemical libraries as apotential binder within the Stat3 SH2 domain and an inhibitor of Stat3activation. S3I-201 shows 2-fold preference for Stat3 over Stat5 andgreater than 3-fold preference over Stat1 (FIGS. 2A-1 and 2A-2, andTable 3).

TABLE 3 IC₅₀ (mM) values for the inhibition of STATs DNA-bindingactivity in vitro NSC # Stat3:3 Stat1:3 Stat1:1 Stat5:5 74859 86 ± 33160 ± 43 >300 166 ± 17 (S3I-201)

Example 3—S3I-201 Disrupts Stat3:Stat3 Complex Formation In Vitro and inIntact Cells

Based on the computational modeling, S3I-201 is predicted to interactwith the SH2 domain of Stat3, thereby inhibiting active Stat3DNA-binding activity (see FIGS. 1A and 1B). To provide experimental datain support of S3I-201's binding to Stat3, the present inventorsinvestigated whether unphosphorylated, inactive Stat3 monomer couldinterfere with the inhibitory effect of S3I-201 on active Stat3DNA-binding (inactive Stat3 monomer will interfere with the inhibitoryactivity of S3I-201 if it interacts with the compound). To make thisdetermination, cell lysates of unphosphorylated, inactive Stat3 monomerprotein prepared from Sf-9 insect cells infected with only baculoviruscontaining Stat3, as previously described (Turkson, J. et al. J. Biol.Chem., 2001, 276:45443-45455; Turkson, J. et al. Mol Cancer Ther, 2004,3:261-269; Turkson, J. et al. Mol. Cancer Ther., 2004, 3:1533-1542;Turkson, J. et al. J Biol Chem., 2005, 280:32979-32988), and celllysates of activated Stat3 dimer protein were mixed together and themixture was pre-incubated with S3I-201 for 30 minutes prior toincubation with the radiolabeled hSIE probe and EMSA analysis, as waspreviously done in FIGS. 2A-1 and 2A-2. The unphosphorylated, inactiveStat3 monomer by itself had no significant effect on DNA-bindingactivity of activated Stat3 (FIG. 2B-1, compare lane 2 to lane 1), asinactive Stat3 monomer is incapable of binding DNA (Turkson, J. et al. JBiol Chem., 2005, 280:32979-32988). Consistent with results in FIGS.2A-1 and 2A-2, pre-incubation of activated Stat3 lysates with 100 μMS3I-201 completely inhibited Stat3 DNA-binding activity (FIG. 2B-1,lanes 3 and 4). By contrast, the presence of inactive Stat3 monomerdiminished the inhibitory effect of S3I-201 on the activated Stat3 in adose-dependent manner, resulting in the recovery of the active Stat3DNA-binding activity (FIG. 2B-1, lanes 5-7). The Stat3 DNA-bindingactivity that was otherwise inhibited (FIG. 2B-1, lanes 3 and 4) waspartially or completely restored in the presence of 4 or 5 μl ofinactive Stat3 lysates, respectively (FIG. 2B-1, lanes 6 and 7).Therefore, while the inactive Stat3 monomer protein is unable to bindDNA, it is capable of interacting with S3I-201 by virtue of its SH2domain. In turn, this interaction reduces the concentration of S3I-201that is available to inhibit the activated Stat3. These findings supportthe S3I-201:Stat3 interaction, which is consistent with the predictionsfrom the computational modeling, and suggest the interaction isindependent of the activation status of Stat3. To determine whetherunphosphorylated, inactive Stat1 or Stat5 monomer or the unrelated Srcprotein (with a SH2 domain) would have effect on S3I-201, similarstudies were performed using independently prepared cell lysates fromSf-9 insect cells infected with only the baculovirus containing eitherStat1, Stat5 or Src, as the present inventors have previously reported(Turkson, J. et al. J. Biol. Chem., 2001, 276:45443-45455; Turkson, J.et al. Mol Cancer Ther, 2004, 3:261-269; Turkson, J. et al. Mol. CancerTher., 2004, 3:1533-1542; Turkson, J. et al. J Biol Chem., 2005,280:32979-32988), and containing either of these proteins. In contrastto the effect observed with the inactive Stat3 monomer (FIG. 2B-1, lanes5 to 7), EMSA analysis shows the presence of inactive Stat1, Stat5, orSrc lysate induces no significant recovery of Stat3 DNA-binding activity(FIG. 2B-1, lanes 8 to 10, 11 to 13, and 14 to 16). In the case of theStat1 monomer lysate, minimal recovery of Stat3 DNA-binding activity isobserved (FIG. 2B-1, lane 10), which is evidence of a weak interactionof the Stat1 protein with S3I-201, as revealed in the initial evaluation(FIG. 2A-2). Compared to the effect of inactive Stat3 monomer lysates,the minimal to no effect of inactive Stat1 or Stat5 monomer, or theunrelated Src lysate suggests selective interaction of Stat3 withS3I-201, presumably through its SH2 domain. The amounts of proteins foreach of Stat1, Stat3, or Stat5 monomer, or the Src lysate used in thesestudies was determined by SDS-PAGE and Western blot analysis to benearly similar (FIG. 2B-2, lanes 17 to 20).

To further confirm the interaction of S3I-201 with Stat3 and todemonstrate that it blocks Stat3:Stat3 dimerization in intact cells,Stat3 pull-down assays involving two differently-tagged Stat3 proteins,FLAG-tagged Stat3 (FLAG-ST3) and Stat3-YFP, expressed in cells wereperformed. Viral Src transformed (NIH3T3/v-Src) mouse fibroblasts stablyexpressing Stat3-YFP were transiently-transfected with FLAG-ST3, andtreated either with 0.05% DMSO (control) or with S3I-201 for 24 hoursand then subjected to pull-down assay using anti-FLAG or anti-YFPantibody and SDS-PAGE. Analysis by Western blot for FLAG of whole-celllysates shows equal expression of the FLAG-ST3 protein in the lysates inthe transiently-transfected cells in both the control (DMSO-treated) andS3I-201-treated cells (FIG. 2C-2). Western blot analysis probing withanti-FLAG antibody of the Stat3-YFP immunoprecipitates shows thepresence of FLAG-ST3 protein in the pulled-down lysate from controlcells (FIG. 2C-1, left panel, upper lane 1), suggesting Stat3-YFP andFLAG-ST3 proteins were pulled down together as a complex. By contrast,Western blot analysis probing with anti-FLAG antibody shows nodetectable level of FLAG-ST3 protein in the Stat3-YFP immunoprecipitatesfrom S3I-201-treated cells (FIG. 2C-1, left panel, upper lane 2 vs. lane1), suggesting the disruption by S3I-201 of the complex formationbetween Stat3-YFP and FLAG-ST3 proteins. The amounts of Stat3-YFP in theimmunoprecipitates are shown (FIG. 2C-1, lower left panel). Similarly,Western blot analysis probing with anti-YFP antibody of the FLAG-ST3immunoprecipitates shows Stat3-YFP present in the pulled-down lysatefrom the 0.05% DMSO-treated (control) cells (FIG. 2C-1, right panel,lower lane 3), but significantly reduced in the FLAG-ST3immunoprecipitates from the S3I-201-treated cells (FIG. 2C-1, rightpanel, lower lane 4 vs. lane 3). These findings suggestFLAG-ST3:Stat3-YFP complex is strongly formed in the control cells, butis significantly diminished in the S3I-201-treated cells. The FLAG-ST3protein amounts in the immunoprecipitates are shown (FIG. 2C-1, upperright panel). Together the findings indicate that S3I-201 disruptsStat3:Stat3 dimers, suggesting that the compound interacts with theStat3 SH2 domain in intact cells.

Example 4—S3I-201 does not Interfere with Lck-SH2 Domain-PhosphotyrosineInteraction

The computational modeling predicts that S3I-201 interacts with theStat3 SH2 domain, thereby inhibiting Stat3 DNA-binding activity (FIGS.1A and 1B). To further investigate the selectivity of S3I-201 and torule out the possibility that it interacts with other SH2domain-containing proteins, the present inventors evaluated its effecton the binding between the unrelated Src family protein, Lck, and thecognate phosphopeptide, EPQpYEEIEL (SEQ ID NO:2) (where pY representspTyr of SEQ ID NO:1). The present inventors used the in vitro ELISAstudy involving the Lck-SH2-GST protein and the conjugate pTyr peptide,biotinyl-ε-Ac-EPQpYEEIEL-OH (SEQ ID NO:2) (Lee, T. R. and Lawrence, D.S. J Med Chem., 2000, 43:1173-1179), as described in “Materials andMethods”. Results from the ELISA show that the co-presence of theLck-SH2-GST protein and its cognate pTyr peptide results in signalinduction (FIG. 2D, bar 4), suggesting an interaction between the two(Lee, T. R. and Lawrence, D. S. J Med Chem., 2000, 43:1173-1179). Theaddition of 30 μM and 100 μM S3I-201 has no effect on the signalinduction (FIG. 2D, compare bars 5 and 6 to the bar 4), indicating thatS3I-201 does not interfere with the binding of the Lck SH2 domain to itscognate pTyr peptide, EPQpYEEIEL (SEQ ID NO:2).

Example 5—S3I-201 Inhibits Stat3 Activation in Intact Cells

It has previously been shown that Stat3 is constitutively-activated in avariety of malignant cells (Yu, C. L. et al. Science, 1995, 269:81-83;Garcia, R. et al. Oncogene, 2001, 20:2499-2513; Turkson, J. et al. Mol.Cell. Biol., 1998, 18:2545-2552). To determine the effect of S3I-201 onintracellular Stat3 activation, NIH3T3/v-Src mouse fibroblasts and humanbreast cancer MDA-MB-231, MDA-MB-435 and MDA-MB-468 cells that harborconstitutively-active Stat3 were treated with the compound and nuclearextracts prepared for Stat3 DNA-binding activity in vitro and EMSAanalysis. Compared to control (0.05% DMSO-treated cells, lane 1),treatment with S3I-201 induced a time-dependent inhibition ofconstitutive Stat3 activation in NIH3T3/v-Src fibroblasts (FIG. 2E,lanes 4-6). By 24 hours, constitutive Stat3 activation was significantlyinhibited in the v-Src-transformed mouse fibroblasts and in the humanbreast cancer MDA-MB-231, MDA-MB-435 and MDA-MB-468 cells (FIG. 2E,lanes 4-6 and 8, 10, and 12). Furthermore, SDS-PAGE and Western blotanalysis of whole-cell lysates from NIH3T3/v-Src fibroblasts showpTyr705 Stat3 levels were significantly diminished following 24-hourtreatment with S3I-201 (FIG. 2F), while total Stat3 protein levelremained unchanged. This inhibition of tyrosine phosphorylation may beexplained by the fact that by binding to the Stat3 SH2 domain, S3I-201prevents Stat3 from binding to the pTyr motifs of the receptor tyrosinekinases (RTKs) and subsequently blocks de novo phosphorylation bytyrosine kinases. To investigate non-specific effects, SDS-PAGE andWestern blot analysis was performed on whole-cell lysates from mousefibroblasts transformed by v-Src (NIH3T3/v-Src) or overexpressing thehuman EGFR (NIH3T3/hEGFR) and stimulated by EGF to determine the abilityto inhibit other signaling proteins. Treatment with S3I-201 for 24 hourshad no significant effect on the phosphorylation of Shc (pShc), Erk1/2(pErk1/2), or Src (pSrc) in cells (FIGS. 2G-1 and 2G-2). Total Erk1/2protein levels were unchanged. Moreover, SDS-PAGE and Western blotanalysis with the anti-pTyr antibody 4G10 clone shows no significantchanges in the pTyr profile of NIH3T3/v-Src fibroblasts following24-hour treatment with S3I-201 (FIG. 2G-4), while same treatmentcondition significantly diminishes pTyr705 Stat3 levels (FIG. 2F). Theseresults together indicate that at the concentrations that inhibit Stat3activity, S3I-201 does not significantly interfere with other signaltransduction mechanisms.

Example 6—Selective Inhibition of Stat3 Transcriptional Activity byS3I-201

The ability of S3I-201 to inhibit Stat3:Stat3 complex formation andStat3 DNA-binding activity prompted us to investigate its effect onStat3-dependent transcriptional activity. Normal mouse fibroblasts(NIH3T3) were transiently co-transfected with the Stat3-dependentluciferase reporter, pLucTKS3 and a plasmid encoding the v-Srconcoprotein that activates Stat3 and cells were untreated (0.05% DMSO,control) or treated with S3I-201. As previously reported (Turkson, J. etal. Mol. Cell. Biol., 1998, 18:2545-2552), v-Src protein mediatedinduction of the Stat3-dependent luciferase reporter, pLucTKS3 activityby about 7-fold (FIG. 3A). This Stat3-dependent pLucTKS3 induction wassignificantly inhibited by the treatment of cells with S3I-201 in adose-dependent manner (FIG. 3A). To examine specificity of effects,normal mouse fibroblasts were co-transfected with the Stat3-independentluciferase reporters, pLucSRE (Turkson, J. et al. Mol. Cell. Biol.,1998, 18:2545-2552) or β-Casein promoter-driven Luc (Galbaugh, T. et al.BMC Cell Biol., 2006, 7:34) together with v-Src plasmid and treated withS3I-201 or without (0.05% DMSO). In contrast to the effect on theinduction of pLucTKS3 luciferase reporter, v-Src-mediated induction ofStat3-independent pLucSRE (Turkson, J. et al. Mol. Cell. Biol., 1998,18:2545-2552) or the β-Casein promoter Luc (Galbaugh, T. et al. BMCCellBiol., 2006, 7:34) was not affected by treatment with S3I-201 (FIGS.3B and 3C). These results demonstrate that S3I-201 selectively inhibitsStat3-dependent transcriptional activity, with no effect onStat3-independent transcriptional events.

Example 7—S3I-201 Blocks Anchorage-Dependent and Independent Growth Onlyin Cells where Stat3 is Persistently Activated

The above results of FIGS. 2 and 3 demonstrate that S3I-201 disruptsStat3 activation. The present inventors next determined whether thisStat3 activity inhibitor is able to inhibit the anchorage-dependent and-independent (transformation) growth of human and mouse cancer celllines and whether this inhibition is dependent on the presence ofpersistently-active Stat3. The human breast carcinoma (MDA-MB-231,MDA-MB-435 and MDA-MB-468) cell lines and the v-Src-transformed mousefibroblasts (NIH3T3/v-Src) that harbor constitutively-active Stat3, andthe human breast carcinoma MDA-MB-453 cell line and normal mousefibroblasts (NIH3T3) that do not harbor aberrant Stat3 activity weretreated with S3I-201 and analyzed for viable cell number by trypan blueexclusion and microscopy (FIG. 4A-4F) or MTT assay (data not shown).Treatment with S3I-201 significantly reduced viable cell numbers andinhibited growth of transformed mouse fibroblasts NIH3T3/v-Src andbreast carcinoma cell lines (MDA-MB-231, MDA-MB-435 and MDA-MB-468)(FIGS. 4E, 4D, and 4F, respectively). By contrast, growth and viabilityof normal mouse fibroblasts (NIH3T3) and breast carcinoma cell line(MDA-MB-453) without aberrant Stat3 activity were not significantlyaltered (FIGS. 4A and 4B). Thus, S3I-201 affected only those cell linesharboring aberrant Stat3, consistent with inhibition of Stat3DNA-binding activity (Table 3 and FIG. 2).

To further examine the effects of S3I-201 on Stat3 biological functions,the compound was tested for its ability to inhibit the growth of v-Srctransformed mouse fibroblasts (NIH3T3/v-Src) in soft-agar suspension incolony formation assays. Results show that growth of v-Src transformedmouse fibroblasts in soft-agar suspension is significantly inhibited byS3I-201 (FIG. 4G). By contrast, soft-agar growth of v-Ras transformedcounterpart (NIH3T3/v-Ras) that is independent of constitutively-activeStat3 is unaffected by treatment with S3I-201 (FIG. 4G), indicating thatS3I-201 selectively inhibits Stat3-mediated malignant transformation.

Example 8—S3I-201 Preferentially Induces Apoptosis of Malignant CellsHarboring Constitutively-Active Stat3

The present inventors next determined if the S3I-201 induced loss oftumor cell viability is due to apoptosis. To this end, the human breastcarcinoma cell lines, MDA-MB-453 and MDA-MB-435, and the normal mousefibroblasts (NIH3T3) and their v-Src transformed counterpart(NIH3T3/v-Src) were untreated (0.05% DMSO, control) or treated withS3I-201 for 48 hours and analyzed by Annexin V binding and FlowCytometry. At 30-100 μM, S3I-201 induced significant apoptosis in therepresentative human breast carcinoma cell line, MDA-MB-435 and theNIH3T3/v-Src, all of which harbor constitutively-active Stat3 (FIG. 5A).The breast carcinoma MDA-MB-435 cell line is more sensitive to 30 μMS3I-201 (FIG. 5A). By contrast, the human breast cancer MDA-MB-453 cellsand the normal mouse fibroblasts (NIH3T3) that do not contain abnormalStat3 activity are less sensitive to S3I-201 at 100 μM or less (FIG.5A). These findings indicate that at concentrations that inhibit Stat3activity, S3I-201 selectively induces apoptosis of transformed cellsharboring aberrant Stat3 signaling, suggesting the inhibition ofconstitutively-active Stat3 is part of the underlying mechanism ofapoptosis by S3I-210. At 300 μM or higher, S3I-201 induced general,non-specific cytotoxicity independent of Stat3 activation status.

The present inventors reasoned that if the ability of S3I-201 to inducetumor cell apoptosis is due to its ability to inhibit Stat3 activation,then the constitutively-dimerized and persistently-activated Stat3C(Bromberg, J. F. et al. Cell, 1999, 98:295-303) should rescue fromS3I-201-induced apoptosis. To this end, the breast carcinoma MDA-MB-231cells that harbor activated Stat3 were transiently transfected withStat3C and evaluated for apoptosis. Twenty-four hours aftertransfection, cells were treated or untreated with S3I-201 for anadditional 24-48 hours, harvested and analyzed by Annexin V binding andFlow Cytometry. Consistent with results in FIG. 5A, S3I-201 induced50-80% apoptosis in untransfected or mock-transfected human breastcarcinoma MDA-MB-231 cells (FIG. 5B, left panel). By contrast, cellstransfected with Stat3C and treated with S3I-201 showed greatlydiminished apoptosis (FIG. 5B, left panel)-less than 2-fold comparedwith 9- or 5-fold apoptosis in non-transfected or mock-transfectedcells, respectively. There is increased in the background level of celldeath, which could be due to the effects of transfection. The rescuefrom the apoptotic effects of S3I-201 can be explained on the basis thatthe artificially-designed activated Stat3C is not inhibited by S3I-201and is sufficient to promote the biological effects of the endogenousStat3. Thus the activated Stat3C rescues cells from the apoptoticeffects of S3I-201 by compensating for the loss of endogenous Stat3activity due to inhibition by S3I-201.

To establish that the effect of S3I-201 is due to its interaction withthe Stat3 SH2 domain, similar studies were performed in cells which weretransiently-transfected with either an expression vector for theN-terminal region of Stat3 (ST3-NT) or the Stat3 SH2 domain (ST3-SH2)and treated with or without S3I-201. Annexin V binding and FlowCytometry showed that while mock-transfected cells were strongly inducedby S3I-201 to undergo apoptosis (FIG. 5B, left panel), similar to theStat3C-transfected cells, the overexpression of the Stat3-SH2 domaindiminished the apoptotic effects of S3I-201 (FIG. 5B, left panel). Incontrast, cells overexpressing the ST3-NT region showed strong inductionof apoptosis (FIG. 5B, left panel), suggesting ST3-NT has no effect onthe ability of S3I-201 to induce apoptosis of malignant cells. Thepresent inventors infer that the exogenous ST3-SH2 domain binds toS3I-201, thereby preventing the compound from binding to and inhibitingendogenous constitutively-active Stat3 protein and its biologicalfunctions.

Example 9—S3I-201 Represses the Expression of the Stat3 Regulated GenesCyclin D1, Bcl-xL, and Survivin

To investigate the molecular mechanisms for the cell growth inhibitionand apoptosis by S3I-201, the present inventors examined the expressionof the known Stat3 target genes in the v-Src-transformed mousefibroblasts (NIH3T3/v-Src) and the human breast carcinoma MDA-MB-231cell line that harbor constitutively-active Stat3. Immunoblot analysisof whole-cell lysates shows significant reduction in expression of theCyclin D1, Bcl-xL and Survivin proteins in response to S3I-201 treatment(FIG. 5C), indicating that S3I-201 represses induction of the cell cycleand anti-apoptotic regulatory genes in malignant cells. These findingsare consistent with the biological effects induced by S3I-201 (FIGS.3-5), and correlate with the inhibition of aberrant Stat3 activity.

Example 10—S3I-201 Induces Regression of Human Breast Tumor Xenografts

The aforementioned findings demonstrate that S3I-201 possesses a stronginhibitory activity against aberrant Stat3, potently inhibitsanchorage-dependent and -independent tumor cell growth, and inducesapoptosis of malignant cells in a Stat3-dependent manner. The presentinventors extended these studies to evaluate the antitumor efficacy ofS3I-201 using mouse models of human breast tumor xenografts that harborconstitutively-active Stat3. Human breast (MDA-MB-231) tumor-bearingmice were given i.v. injection of S3I-201 or vehicle every 2 or every 3days for two weeks, and tumor measurements were taken every 2 to 3 days.Compared to control (vehicle-treated) tumors, which continued to grow,strong growth inhibition of human breast tumors were observed in micethat received S3I-201 (FIG. 6A). Continued evaluation of treated miceupon termination of treatment showed no resumption of tumor growth (datanot shown), suggesting potentially a long-lasting effect of S3I-201 ontumor growth. To determine that target was inhibited by S3I-201, lysateswere prepared from tumor tissue from one control animal and from theresidual tumor tissue in two treated-mice for Stat3 DNA-binding activityin vitro, as the present inventors have previously done (Turkson, J. etal. Mol. Cancer Ther., 2004, 3:1533-1542). EMSA analysis showed stronginhibition of Stat3 DNA binding activity in residual tumor tissue frommice treated with S3I-201 (T1 and T2) compared to control tumor (FIG.6B, lanes 1 to 3). SDS-PAGE and Western blot analysis of the lysatesrevealed minimal to no detection of pTyr Stat3 (pYStat3) in residualtumor tissue from treated mice (T1 and T2) compared to control (FIG. 6C,upper panel). Total Stat3 protein remained unchanged (FIG. 6C, lowerpanel). Moreover, EMSA analysis of in vitro Stat3 DNA-binding activityalso shows that pre-incubation of lysates of equal total protein fromcontrol tumor tissue with 10, 30, and 100 μM S3I-201 prior to incubationwith radiolabeled hSIE probe resulted in a dose-dependent abrogation ofStat3 activity (FIG. 6B, lanes 4 to 6), as observed originally in FIG.2A-1. Together, these studies establish the proof-of-concept for theantitumor effect of S3I-201 in tumors that harbor constitutively-activeStat3.

The data in the Examples demonstrate the feasibility of using X-raycrystallographic data and computational modeling as a basis for astructure-based virtual screening to identify chemical probes andsmall-molecule binders of the Stat3 SH2 domain. The docking approachwith GLIDE 2.7 (Friesner, R. et al. J. Med. Chem., 2004, 47:1739-1749;Halgren, T. et al. J. Med. Chem., 2004, 47:1750-1759) was firstvalidated using the native pTyr peptide, APY*LKT, which produced adocking mode that closely resembled the X-ray crystal structure (Becker,S. et al. Nature, 1998, 394:145-151). By this approach, the presentinventors have docked and scored small-molecules from the NCI Chemicallibraries into the Stat3 SH2 domain, and identified potent binders thatranked within the top ˜0.1% of the docked and scored Plated Set 3Dstructures. S3I-201 (NSC 74859) emerged as a potent inhibitor of Stat3DNA-binding activity in vitro. Similar computational analysis has beenapplied in the development of inhibitors of Stat3 and other proteins(Shao, H. et al. J Biol Chem., 2004, 279:18967-18973; Song, H. et al.Proc Natl Acad Sci USA, 2005, 102:4700-4705). The docking pose ofS3I-201 suggests that its aminosalicylic moiety mimics thephenylphosphate group of the PY*LKT motif. Furthermore, its carboxylgroup is hydrogen bonded to the Arg 609, Ser 611 and Ser 613 of Stat3,while the phenolic hydroxyl group is hydrogen-bonded to Lys 591.Moreover, the proximity of the Lys 591 to the benzene ring that containsthe phenolic hydroxyl group suggests the possibility of a stabilizingpi-cation interaction. Also, the tolyl group is buried within apartially hydrophobic pocket, which contains the tetramethylene portionof the side chain of Lys 592 and the trimethylene portion of the sidechain of Arg 595, along with residues Ile 597 and Ile 634. The presentinventors believe that these features (the phosphate mimic andhydrophobic side chain) combine and explain how S3I-201 binds to the SH2domain of Stat3.

The native pTyr peptide inhibitor of Stat3, APY*LKT (SEQ ID NO:1), waspreviously shown to disrupt Stat3:Stat3 dimerization, thereby inducingantitumor cell effects (Turkson, J. et al. Mol Cancer Ther, 2004,3:261-269). The computational analysis predicts that S3I-201 is areasonably strong binder within the Stat3 SH2 domain, raising thepossibility that it might disrupt Stat3:Stat3 dimerization as a mode ofinhibition of Stat3. Using differently-tagged Stat3 monomer proteins inpull-down assays, the present inventors demonstrate that S3I-201disrupts the complex formation between two Stat3 monomers. S3I-201preferentially interacts with Stat3 monomer protein over Stat1 or theSrc family protein, and has a 2-fold lower potency for Stat5. In cells,the interactions with Stat3 will prevent binding of the Stat3 SH2 toRTKs and Src and/or Jaks and inhibit de novo phosphorylation andactivation of Stat3 monomer. Thus, S3I-201 mediates selective inhibitionof Stat3 transcriptional activity, induces cell growth inhibition, lossof viability, and apoptosis of human breast cancer and v-Src-transformedmouse cells that harbor constitutively-active Stat3, as well as blocksv-Src transformation, events which are all consistent with theabrogation of Stat3 activation (Turkson, J. et al. J. Biol. Chem., 2001,276:45443-45455; Turkson, J. et al. Mol Cancer Ther, 2004, 3:261-269;Garcia, R. et al. Oncogene, 2001, 20:2499-2513; Catlett-Falcone, R. etal. Immunity, 1999, 10:105-115; Mora, L. B. et al. Cancer Res, 2002,62:6659-6666). The observation that overexpressed exogenous Stat3 SH2domain, but not Stat3 N-terminus in malignant cells protects againstS3I-201-induced apoptosis strongly supports the Stat3 SH2 domain as thetarget of S3I-201, and for that reason will bind to S3I-201 in cells,thereby preventing the S3I-201 from inhibiting endogenous Stat3activity. Moreover, the rescue from the apoptotic effects of S3I-201 bythe ectopic expression of the constitutively-active Stat3C in malignantcells demonstrates that Stat3C compensates for the absence of aberrantStat3 signaling. Thus, these findings further strongly suggest theanti-tumor cell effects of S3I-201 are mediated by suppressing aberrantStat3-induced dysregulation of the cell cycle control and theanti-apoptotic genes. Furthermore, in vivo antitumor effects of S3I-201in human breast tumor xenografts establish the proof-of-concept for thetherapeutic potential of S3I-201 as a Stat3 inhibitor in human tumorsthat harbor constitutively-active Stat3.

Described herein is the first report of a Stat3 SH2 domain binder and aninhibitor of Stat3 signaling, which is structurally different frompreviously identified dimerization disruptors, peptides andpeptidomimetics (Turkson, J. et al. J. Biol. Chem., 2001,276:45443-45455; Turkson, J. et al. Mol Cancer Ther, 2004, 3:261-269),or other Stat3 inhibitors, such as the g-quartet oligonucleotides (Jing,N. et al. DNA Cell Biol, 2003, 22:685-696), peptide aptamers(Nagel-Wolfrum, K. et al. Mol Cancer Res, 2004, 2:170-182), platinum(IV) complexes (Turkson, J. et al. Mol. Cancer Ther., 2004, 3:1533-1542;Turkson, J. et al. J Biol Chem., 2005, 280:32979-32988), cucurbitacin(Blaskovich, M. A. et al. Cancer Res, 2003, 63:1270-1279), and STA-21(NSC 628869) (Song, H. et al. Proc Natl Acad Sci USA, 2005,102:4700-4705). While SH2 domains have been noted to have a highsequence similarity in their 100 amino acids (Sheinerman, F. B. et al.JMol Biol., 2003, 334:823-841; Kuriyan, J. and Cowburn, D. Annu RevBiophys Biomol Struct., 1997, 26:259-288), S3I-201 has preference forStat3 in that at concentrations that inhibit Stat3, S3I-201 has loweffect on Stat1 and Stat5, no interaction with the Src family proteins,weak inhibition of Erks 1/2 and Shc activation, and low toxicity tocells with no aberrant Stat3.

The study described herein supports computational modeling applicationin structure-based virtual screening for identifying Stat3 inhibitorsfrom chemical libraries, and together with another report (Song, H. etal. Proc Natl Acad Sci USA, 2005, 102:4700-4705) is among the first toidentify Stat3 inhibitors by this approach. S3I-201 represents a newlead for developing combinatorial libraries with increased diversity,thereby setting a new course in the Stat3 inhibitor design.

Example 11—Compound Synthesis

The following compounds were prepared according to Scheme 1

Ethyl 2-(tosyloxy)acetate (2). A solution of ethyl glycolate (500 mg,4.8 mmol) and tosyl chloride (915 mg, 4.8 mmol) in anhydrous diethylether (2 ml) at 0° C. was treated dropwise with triethylamine (1.34 ml,9.6 mmol). The temperature was maintained at 0° C. with stirring for afurther 2 h. After this time, water was added and the phases separated.The aqueous phase was extracted with fresh diethyl ether. The combinedorganic extracts were dried (MgSO₄), filtered and the solvent removed invacuo. The crude product was purified by column chromatography oversilica gel (R_(f)=0.29, 25% ethyl acetate in hexane) affording thetosylate 2 as a colorless oil which solidified on standing as a whitesolid (868 mg, 70%). [This procedure was repeated on 2×scale (1.8 g,73%)]. δ_(H) (400 MHz, CDCl₃) 1.22-1.27 (3H, m, CH₃), 2.46 (3H, s,ArCH₃), 4.16-4.22 (2H, m, CH ₂CH₃), 4.58-4.59 (2H, m, OCH₂), 7.36 (2H,d, J=8.2 Hz, ArH), 7.84 (2H, d, J=8.2 Hz, ArH); δ_(C) (100.6 MHz, CDCl₃)14.2, 21.9, 62.1, 64.9, 128.3, 130.1, 132.9, 145.5, 166.2; MS (ES⁺)276.0 (100%, [M+H₂O]⁺) (ion not detected in ES⁻).

2-(Tosyloxy)acetic acid (3). A solution of ethyl 2-(tosyloxy)acetate (2,860 mg, 3.33 mmol) in ethanol (4 ml) was treated at room temperaturewith 5% NaOH aqueous solution (2 ml). The resulting mixture was stirredat room temperature for 3 h. The ethanol was removed in vacuo andaqueous 5% HCl solution (2.7 ml) was added. The aqueous mixture wasextracted with chloroform and the organic extracts dried (Na₂SO₄),filtered and the solvent removed under reduced pressure. The remainingwhite solid was recrystallized from hexane/ethyl acetate as a whitepowder (476 mg, 62%). [This reaction was repeated on 2× scale (1.449 g,94%)]. δ_(H) (400 MHz, MeOD) 2.45 (3H, s, Me), 4.58 (2H, s, CH₂), 7.44(2H, d, J=8.2 Hz, ArH), 7.82 (2H, d, J=8.2 Hz, ArH); δ_(C) (100.6 MHz,MeOD) 20.4, 64.7, 128.0, 129.9, 133.0, 145.6, 168.2; MS (ES⁺) 247.8(100%, [M+H₂O]⁺); MS (ES⁻) 170.1 (100%, TsO⁻).

2-Chloro-2-oxoethyl-4-methylbenzenesulfonate (4). A mixture of2-(tosyloxy)acetic acid (3, 400 mg, 1.74 mmol) and thionyl chloride (1ml) was heated at reflux for 1.5 h. The excess thionyl chloride wasremoved in vacuo and the resulting white solid dried under high vacuumat 50° C. for 1.5 h. The product acid chloride was used without furtherpurification.

2-Hydroxy-4-(2-(tosyloxy)acetamido)benzoic acid 1 (NSC-59263). A mixtureof p-amino salicylic acid (102 mg, 0.67 mmol) and NaOH (27 mg, 0.67mmol) in water (6 ml) was stirred at room temperature for 10 min untilall of the solid material had dissolved. Na₂CO₃ (59 mg, 0.5561 mmol) wasthen added and the mixture cooled to 0° C. A solution of2-chloro-2-oxoethyl-4-methylbenzenesulfonate (4, 200 mg, 0.8044 mmol) inTHF (2 ml) was injected rapidly and the resulting solution allowed towarm to room temperature and stirred for 2 h. Then reaction mixture waspoured into a separating funnel containing diethyl ether. The phaseswere separated and the aqueous phase washed with a further portion ofdiethyl ether. The aqueous phase was acidified to pH 1 by addition of 1NHCl solution and the product extracted into ethyl acetate. The ethylacetate was washed twice with 1N HCl solution and the organic phase wasdried (Na₂SO₄), filtered and the solvent removed in vacuo. The resultingpale yellow solid was triturated with chloroform leaving 1 as a whitepowder (155 mg, 63%). δ_(H) (400 MHz, DMSO) 2.37 (3H, s, Me), 4.68 (2H,s, CH₂), 6.93 (1H, d, J=7.4 Hz, ArH), 7.17 (1H, s, ArH), 7.46 (2H, d,J=6.8 Hz, ArH), 7.69 (1H, d, J=7.4 Hz, ArH), 7.81 (2H, d, J=6.8 Hz,ArH), 10.32 (1H, s, NH); δ_(C) (100.6 MHz, DMSO) 21.8, 68.0, 107.0,110.7, 128.5, 130.9, 131.7, 132.7, 144.5, 146.0, 162.8, 164.5, 172.2; MS(ES⁺) 366.0 (100%, [M+H]⁺), 388.0 (50%, [M+Na]⁺); (ion not detected inES⁻).

4-(2-(Tosyloxy)acetamido)benzoic acid (5). A mixture of p-aminobenzoicacid (92 mg, 0.67 mmol) and NaOH (27 mg, 0.67 mmol) in water (6 ml) wasstirred at room temperature for 10 min until all of the solid materialhad dissolved. Na₂CO₃ (59 mg, 0.5561 mmol) was then added and themixture cooled to 0° C. A solution of2-chloro-2-oxoethyl-4-methylbenzenesulfonate (4, 200 mg, 0.8044 mmol) inTHF (2 ml) was injected rapidly and the resulting solution allowed towarm to room temperature and stirred for 2 h. Then reaction mixture waspoured into a separating funnel containing diethyl ether. The phaseswere separated and the aqueous phase washed with a further portion ofdiethyl ether. The aqueous phase was acidified to pH 1 by addition of 1NHCl solution and the product extracted into ethyl acetate. The ethylacetate was washed twice with 1N HCl solution and the organic phase wasdried (Na₂SO₄), filtered and the solvent removed in vacuo. The resultingpale orange solid was recrystallized from chloroform/MeOH (20:1)affording 5 as a white powder (146 mg, 62%). δ_(H) (400 MHz, DMSO) 2.37(3H, s, Me), 4.70 (2H, s, CH₂), 7.46 (2H, d, J=8 Hz, ArH), 7.59 (2H, d,J=8.6 Hz, ArH), 7.82 (2H, d, J=8 Hz, ArH), 7.87 (2H, d, J=8.6 Hz, ArH),10.39 (1H, s, NH), 12.72 (1H, CO₂H); δ_(C) (100.6 MHz, DMSO) 21.8, 68.0,119.4, 126.4, 128.5, 130.9, 131.1, 132.7, 142.7, 146.0, 164.4, 167.5; MS(ES⁺) 349.7 (100%, [M+H]⁺), 371.7 (40%, [M+Na]⁺); MS (ES⁻) 347.9 (100%,[M−H]⁻); HRMS [M−H]⁻ requires 348.05473, found 348.05334.

Compounds 6, 7, and 8

General Procedure: A suspension of the aniline derivative (0.455 mmol)in water (6 ml) was stirred at room temperature for 10 min. Na₂CO₃ (40mg, 0.455 mmol) was then added and the mixture cooled to 0° C. Asolution of 2-chloro-2-oxoethyl-4-methylbenzenesulfonate (4, 125 mg, 0.5mmol) in THF (2 ml) was injected rapidly and the resulting solutionallowed to warm to room temperature and stirred for 2 h.

2-Oxo-2-(phenylamino)ethyl 4-methylbenzenesulfonate (6): A whiteprecipitate formed in the reaction mixture. This precipitate wascollected by filtration, washed with water and dried under high vacuum.This afforded 6 as an off white powder (103 mg, 74%). An analyticalsample was obtained by recrystallization from hexane/ethyl acetate(white needles). δ_(H) (400 MHz, MeOD) 2.40 (3H, s, CH₃), 4.63 (2H, s,CH₂), 7.10 (1H, t, J=7.6 Hz, ArH), 7.28 (2H, t, J=7.6 Hz, ArH),7.41-7.50 (4H, m, ArH), 7.85 (2H, d, J=8 Hz); δ_(C) (100.6 MHz, MeOD)20.4, 67.2, 120.5, 124.7, 128.1, 128.6, 130.0, 132.6, 137.5, 145.9,164.6; MS (ES⁺) 306.0 (100%, [M+H]⁺); HRMS [M+H]⁺ requires 306.07946,found 306.07944.

2-(4-Cyanophenylamino)-2-oxoethyl 4-methylbenzenesulfonate (7): A orangeprecipitate formed in the reaction mixture. This precipitate wascollected by filtration, washed with water and dried under high vacuum.The solid was shown by NMR analysis to comprise of the desired productand starting material. The solid was purified by column chromatography(R_(f)=0.39, 50% ethyl acetate in hexane) affording 7 as an off whitepowder (58 mg, 39%). δ_(H) (400 MHz, MeOD/DMSO spike) 2.41 (3H, s, CH₃),4.68 (2H, s, CH₂), 7.44 (2H, d, J=8 Hz, ArH), 7.66-7.71 (4H, m, ArH),7.86 (2H, d, J=8 Hz, ArH); δ_(C) (100.6 MHz, MeOD/DMSO spike) 20.5,67.3, 107.1, 118.6, 120.1, 128.2, 130.1, 133.1, 142.2, 146.0, 165.0; MS(ES⁺) 330.7 (55%, [M+H]⁺), 347.7 (100%, [M+H₂O]⁺).

2-Oxo-2-(4-sulfamoylphenylamino)ethyl 4-methylbenzenesulfonate (8): Awhite precipitate formed in the reaction mixture. This precipitate wascollected by filtration, washed with water and dried under high vacuum.This afforded 8 as an off white powder (109 mg, 62%). An analyticalsample was obtained by recrystallization from acetone/ethyl acetate(colourless/white crystals). δ_(H) (400 MHz, MeOD/DMSO spike) 2.42 (3H,s, CH₃), 4.68 (2H, s, CH₂), 7.44 (2H, d, J=8 Hz, ArH), 7.67 (2H, d,J=8.4 Hz, ArH), 7.83 (2H, d, J=8.4 Hz, ArH), 7.87 (2H, d, J=8 Hz, ArH);δ_(C) (100.6 MHz, MeOD/DMSO spike) 20.5, 67.3, 119.8, 127.0, 128.2,130.1, 132.7, 139.3, 141.2, 146.0, 164.9; MS (ES⁺) 348.0 (100%), 385.0(20%, [M+H]⁺), 402.0 (50%, [M+H₂O]⁺).

The following compounds were prepared according to Scheme 2.

2-(4-Methylphenylsulfonamido)acetic acid (9). Glycine (1 g, 13.32 mmol)was dissolved in 1N NaOH (aq.) and a solution of tosyl chloride (2.62 g,13.72 mmol) in diethyl ether (15 ml) was added portion wise withstirring over 10 min. The resulting mixture was allowed to stir at roomtemperature for 3 h. The ethereal layer was separated and the aqueouslayer treated with 2N HCl solution until acidified to pH 5. Aftercooling the resulting solution to 0° C., the product began toprecipitate from the solution. The solid was collected by filtration andthe mother liquor placed in a fridge overnight causing more of thedesired product to precipitate. The solid was again collected byfiltration and the combined collected solid was dried under high vacuumaffording 9 as a white powder (613 mg, 21%). δ_(H) (400 MHz, MeOD) 2.41(3H, s, CH₃), 3.66 (2H, s, CH₂), 7.35 (2H, d, J=8 Hz, ArH), 7.73 (2H, d,J=8 Hz, ArH); δ_(C) (100.6 MHz, MeOD) 20.3, 43.6, 127.0, 129.5, 137.6,143.6, 171.0; MS (ES⁺) 246.8 (100%, [M+H₂O]⁺); MS (ES⁻) 227.9 (100%,[M−H]⁻); HRMS [M−H]⁻ requires 228.03360, found 228.03364.

2-(4-Methylphenylsulfonamido)acetyl chloride (10). A mixture of2-(4-methylphenylsulfonamido)acetic acid (9, 200 mg, 0.873 mmol) andthionyl chloride (1 ml) was heated at reflux for 1.5 h. The excessthionyl chloride was removed in vacuo and the resulting white soliddried under high vacuum at 50° C. for 1.5 h. The product acid chloride10 was used without further purification.

2-Hydroxy-4-[4-methylphenylsulfonamido)acetamido]benzoic acid (11). Amixture of p-aminosalicylic acid (111 mg, 0.728 mmol) and NaOH (29 mg,0.728 mmol) in water (6 ml) was stirred at room temperature for 10 minuntil all of the solid material had dissolved. Na₂CO₃ (64 mg, 0.604mmol) was then added and the mixture cooled to 0° C. A solution of2-(4-methylphenylsulfonamido)acetyl chloride (10, 216 mg, 0.873 mmol) inTHF (2 ml) was injected rapidly and the resulting solution allowed towarm to room temperature and stirred for 2 h. Then reaction mixture waspoured into a separating funnel containing diethyl ether. The phaseswere separated and the aqueous phase washed with a further portion ofdiethyl ether. The aqueous phase was acidified to pH 1 by addition of 1NHCl solution and the product extracted into ethyl acetate. The ethylacetate was washed twice with 1N HCl solution and the organic phase wasdried (Na₂SO₄), filtered and the solvent removed in vacuo. The resultingpale yellow solid was triturated with chloroform leaving 11 as an offwhite powder (187 mg, 71%). δ_(H) (400 MHz, MeOD) 2.36 (3H, s, CH₃),3.72 (2H, s, CH₃), 6.90 (1H, dd, J=8.6 Hz and 1.8 Hz, ArH), 7.17 (1H, d,J=1.6, ArH), 7.33 (2H, d, J=8 Hz, ArH), 7.73-7.76 (3H, m, ArH); δ_(C)(100.6 MHz, MeOD) 21.6, 46.6, 106.7, 110.9, 127.3, 130.2, 131.7, 138.1,143.4, 145.5, 162.7, 167.7, 172.2; MS (ES⁺) 364.7 (100%, [M+H]⁺), (ionnot detected in ES⁻).

Ethyl 2-(4′-chlorobiphenyl-4-ylsulfonyloxy)acetate (12). A suspension ofethyl glycolate (73 mg, 0.6964 mmol) and4-chloro[1,1′-biphenyl]-4-sulfonyl chloride (200 mg, 0.6964 mmol) inanhydrous diethyl ether (1 ml) at 0° C. was treated dropwise withtriethylamine (0.194 ml, 1.393 mmol). The temperature was maintained at0° C. with stirring for a further 2 h. After this time, water was addedand the phases separated. The aqueous phase was extracted with freshdiethyl ether. The combined organic extracts were dried (MgSO₄),filtered and the solvent removed in vacuo. The crude product waspurified by column chromatography over silica gel (R_(f)=0.45, 25% ethylacetate in hexane) affording 12 as a colorless oil which solidified onstanding as an off white solid (213 mg, 86%). δ_(H) (400 MHz, CDCl₃)1.25 (3H, t, J=7.2 Hz, CH₃), 4.21 (2H, q, J=7.2 Hz, CH₂), 4.65 (2H, s,CH₂), 7.46-7.55 (4H, m, ArH), 7.73 (2H, d, J=8.8 Hz, ArH), 8.03 (2H, d,J=8.4 Hz, ArH); δ_(C) (100.6 MHz, CDCl₃) 14.2, 62.2, 65.0, 127.9, 128.8,129.0, 129.6, 134.8, 135.4, 137.6, 146.1, 166.2; MS (ES⁺) 372.0 (100%,[M+H₂O]⁺); MS (ES⁻) 266.9 (100%, [M−H]⁻).

2-(4′-Chlorobiphenyl-4-ylsulfonyloxy)acetic acid (13). A solution ofester 12 (200 mg, 0.5633 mmol) in ethanol (0.7 ml) was treated at roomtemperature with 5% NaOH aqueous solution (0.45 ml). The resultingmixture was stirred at room temperature for 3 h. The ethanol was removedin vacuo and aqueous 5% HCl solution (0.46 ml) was added. The aqueousmixture was extracted with chloroform and the organic extracts dried(Na₂SO₄), filtered and the solvent removed under reduced pressure. Theremaining white solid was recrystallized from chloroform as a whitepowder (133 mg, 72%). (Material that did not crystallize was collectedby column chromatography using a gradient ethyl acetate to 50% MeOH inethyl acetate). δ_(H) (400 MHz, MeOD) 4.65 (2H, s, CH₂), 7.50 (2H, d,J=8.4 Hz, ArH), 7.70 (2H, d, J=8.4 Hz, ArH), 7.88 (2H, d, J=8.4 Hz,ArH), 8.02 (2H, d, J=8.4 Hz, ArH); δ_(C) (100.6 MHz, MeOD) δ5.2, 127.7,128.6, 128.8, 129.1, 134.9, 135.0, 137.7, 145.7, 168.3; MS (ES⁺) 343.7(100%, [M+H₂O]⁺), 323.7 (5%, [M+H]⁺), (ion not detected in ES⁻).

2-Chloro-2-oxoethyl 4′-chlorobiphenyl-4-sulfonate (14). A mixture ofacid 13 (100 mg, 0.306 mmol) and thionyl chloride (1 ml) was heated atreflux for 1.5 h. The excess thionyl chloride was removed in vacuo andthe resulting pale yellow/white solid dried under high vacuum at 50° C.for 1.5 h. The product acid chloride 14 was used without furtherpurification.

4-(2-(4′-Chlorobiphenyl-4-ylsulfonyloxy)acetamido)-2-hydroxybenzoic acid(15). A mixture of p-aminosalicylic acid (43 mg, 0.278 mmol) and NaOH(11 mg, 0.278 mmol) in water (6 ml) was stirred at room temperature for10 min until all of the solid material had dissolved. Na₂CO₃ (25 mg,0.231 mmol) was then added and the mixture cooled to 0° C. A suspensionof 14 (105 mg, 0.306 mmol) in THF (2 ml) was injected rapidly and theresulting solution allowed to warm to room temperature and stirred for 2h. Then reaction mixture was poured into a separating funnel containingdiethyl ether. The phases were separated and the aqueous phase washedwith a further portion of diethyl ether. The aqueous phase was acidifiedto pH 1 by addition of 1N HCl solution and the product extracted intoethyl acetate. The ethyl acetate was washed twice with 1N HCl solutionand the organic phase was dried (Na₂SO₄) and filtered. The product wasfound to be in both the diethyl ether and ethyl acetate fractions sothese organic phases were combined and the solvent removed in vacuo. Theproduct was purified by column chromatography over silica gel(R_(f)=0.27, 10% MeOH in ethyl acetate) affording 15 as a yellow powder(45 mg, 35%). An analytical sample was obtained by further purificationby recrystallization from EtOAc/MeOH. δ_(H) (400 MHz, MeOD) 4.72 (2H, s,CH₂), 6.89 (1H, dd, J=8.6 Hz and 1.8 Hz, ArH), 7.13 (1H, d, J=1.8 Hz,ArH), 7.46 (2H, d, J=8.4 Hz, ArH), 7.58 (2H, d, J=8.4 Hz, ArH), 7.70(1H, d, J=8.6 Hz, ArH), 7.82 (2H, d, J=8.4 Hz, ArH), 8.03 (2H, d, J=8.4Hz, ArH); MS (ES⁻) 401.9 (100%), 459.8 (75%, [M−H]⁻).

Ethyl 2-(biphenyl-4-ylsulfonyloxy)acetate (16). A suspension of ethylglycolate (374 μl, 3.957 mmol) and biphenyl-4-sulfonyl chloride (1 g,3.957 mmol) in anhydrous diethyl ether (2 ml) at 0° C. was treateddropwise with triethylamine (1.1 ml, 7.914 mmol). The temperature wasmaintained at 0° C. with stirring for a further 2 h. After this time,water was added and the phases separated. The aqueous phase wasextracted with fresh diethyl ether. The combined organic extracts weredried (MgSO₄), filtered and the solvent removed in vacuo. The crudeproduct was purified by column chromatography over silica gel(R_(f)=0.26, 25% ethyl acetate in hexane) affording 16 as a colourlessoil (1.055 g, 83%). δ_(H) (400 MHz, CDCl₃) 1.25 (3H, t, J=6.8 Hz, CH₃),4.20 (2H, q, J=6.8 Hz, CH₂), 4.65 (2H, s, CH₂), 7.44-7.51 (3H, m, ArH),7.61 (2H, d, J=7.6 Hz, ArH), 7.76 (2H, d, J=8 Hz, ArH), 8.01 (2H, d, J=8Hz, ArH); δ_(C) (100.6 MHz, CDCl₃) 14.2, 62.2, 65.0, 127.6, 128.1,128.9, 129.0, 129.4, 134.4, 139.2, 147.4, 166.2; MS (ES⁺) 338.1 (100%,[M+H₂O]⁺).

2-(Biphenyl-4-ylsulfonyloxy)acetic acid (17). A solution of ester 16(1.05 g, 3.281 mmol) in ethanol (4 ml) was treated at room temperaturewith 5% NaOH aqueous solution (2.6 ml). The resulting mixture wasstirred at room temperature for 3 h. The ethanol was removed in vacuoand aqueous 5% HCl solution (2.6 ml) was added. The aqueous mixture wasextracted with chloroform and the organic extracts dried (Na₂SO₄),filtered and the solvent removed under reduced pressure. The remainingwhite solid was recrystallized from hexane/ethyl acetate as a whitepowder (806 mg, 84%). δ_(H) (400 MHz, MeOD) 4.66 (2H, s, CH₂), 7.41-7.51(3H, m, ArH), 7.69 (2H, d, J=7.2 Hz, ArH), 7.87 (2H, d, J=8.4 Hz, ArH),8.01 (2H, d, J=8.4 Hz, ArH); δ_(C) (100.6 MHz, MeOD) δ4.9, 127.2, 127.7,128.6, 128.7, 129.0, 134.5, 139.1, 147.2, 168.2.

2-Chloro-2-oxoethyl biphenyl-4-sulfonate (18). A mixture of acid 17 (200mg, 0.685 mmol) and thionyl chloride (1 ml) was heated at reflux for 1.5h. The excess thionyl chloride was removed in vacuo and the residuedried under high vacuum at 50° C. for 1.5 h. The product acid chloride18 was used without further purification.

4-(2-(biphenyl-4-ylsulfonamido)acetamido)-2-hydroxybenzoic acid (19). Amixture of p-aminosalicylic acid (95 mg, 0.623 mmol) and NaOH (25 mg,0.623 mmol) in water (6 ml) was stirred at room temperature for 10 minuntil all of the solid material had dissolved. Na₂CO₃ (55 mg, 0.517mmol) was then added and the mixture cooled to 0° C. A suspension of 18(213 mg, 0.685 mmol) in THF (2 ml) was injected rapidly and theresulting solution allowed to warm to room temperature and stirred for 2h. Then reaction mixture was poured into a separating funnel containingdiethyl ether. The phases were separated and the aqueous phase washedwith a further portion of diethyl ether. The aqueous phase was acidifiedto pH 1 by addition of 1N HCl solution and the product extracted intoethyl acetate. The ethyl acetate was washed twice with 1N HCl solutionand the organic phase was dried (Na₂SO₄), filtered and the solventremoved in vacuo. The product was found to be in both the diethyl etherand ethyl acetate fractions so these organic phases were combined andthe solvent removed in vacuo. The product was purified by columnchromatography over silica gel (R_(f)=0.22, 10% MeOH in ethyl acetate)affording 19 as a yellow powder (109 mg, 41%). An analytical sample wasobtained by further purification by recrystallization from EtOAc/hexane.δ_(H) (400 MHz, DMSO) 4.76 (2H, s, CH₂), 6.96 (1H, d, J=8.4 Hz, ArH),7.22 (1H, s, ArH), 7.42-7.74 (6H, m, ArH), 7.93-8.01 (4H, m, ArH), 10.37(1H, s, NH); MS (ES⁺) 428.0 (100%, [M+H]⁺).

4-(2-(Biphenyl-4-ylsulfonyloxy)acetamido)benzoic acid (20). A mixture ofp-aminobenzoic acid (43 mg, 0.31 mmol) and NaOH (12.5 mg, 0.31 mmol) inwater (3 ml) was stirred at room temperature for 10 min until all of thesolid material had dissolved. Na₂CO₃ (27 mg, 0.258 mmol) was then addedand the mixture cooled to 0° C. A suspension of sulfonyl chloride 18(106 mg, 0.341 mmol) in THF (1 ml) was injected rapidly and theresulting solution allowed to warm to room temperature and stirred for 2h. Then reaction mixture was poured into a separating funnel containingdiethyl ether. The phases were separated and the aqueous phase washedwith a further portion of diethyl ether. The aqueous phase was acidifiedto pH 1 by addition of 1N HCl solution and the product extracted intoethyl acetate. The ethyl acetate was washed twice with 1N HCl solutionand the organic phase was dried (Na₂SO₄), filtered and the solventremoved in vacuo. The resulting solid was recrystallized fromhexane/ethyl acetate affording acid 20 as an off white powder (81 mg,64%). δ_(H) (400 MHz, DMSO) 4.79 (2H, s, CH₂), 7.45-7.70 (9H, m, ArH),7.86-8.03 (4H, m, ArH), 10.43 (1H, NH); δ_(C) (100.6 MHz, DMSO) 68.2,119.4, 126.5, 127.9, 128.6, 129.2, 129.6, 129.8, 131.1, 134.3, 138.7,142.7, 146.7, 164.4, 167.5; MS (ES⁺) 411.7 (100%, [M+H]⁺).

The following compounds were prepared according to similar method asshown in Scheme 3

32 and 31 were synthesized as outlined in the Scheme 3

Synthesis of the Intermediate 1a:

The sulfonyl chloride (0.995 g, 3.94 mmol) was suspended in pyridine:DCM(10 ml: 5 ml) and ethyl glycinate (0.500 g, 3.58 mmol) was added at roomtemperature under argon atmosphere, and stirred at room temperature for4 h. The reaction mixture was diluted with EtOAc and washed with 4M HClfollowed by brine. The organic phase was dried (MgSO₄) and concentratedto obtain the intermediate 1a (0.941 g). This intermediate was carriedto the next stage without purification.

The Intermediate 1b was Synthesized Using the Same Procedure.

Synthesis of the Intermediate 2a:

The intermediate 1a (900 mg, 2.82 mmol) was suspended in EtOH:H₂O (9ml:1 ml) and NaOH (5% wt, 50 mg) was added to the reaction mixture andrefluxed for 3h. The reaction mixture was cooled to room temperature,acidified (pH=2-3) and extracted with EtOAc. The combined organic phasewas dried (MgSO₄) and concentrated to obtain the required acid (770 mg)as a white powder. The carboxylic acid obtained was refluxed withthionyl chloride:DCM (3:1) for 2-3 h. The excess thionyl chloride wasremoved under vacuum to give the intermediate 2a (820 mg). This productwas carried to the next stage.

The Intermediate 2b was Synthesized Using the Same Procedure.

4-[2-{4-(Phenyl)phenylsulfonamido)}acetamido]benzoic acid (32).para-Aminosalicylic acid (54 mg, 0.35 mmol) was suspended in water (4ml) and 3 drops of THF was added to dissolve the compound. Na₂CO₃ wasadded to the reaction mixture and cooled to 0° C. A solution ofintermediate 2a (100 mg, 0.322 mmol) in THF (1.5 ml) was added over 15min. The reaction mixture was stirred at room temperature for 3 h. Asolution of 4M HCl was added portionwise to obtain a precipitate. Thewhite precipitate was filtered and washed with water and dried undervacuum to obtain 32 (45 mg) as a pure compound: ¹H NMR (DMSO-d₆, 400MHz) δ 12.7 (broad s, 1H), 8.17 (t, J=12.0 Hz, 1H), 7.88-7.81 (m, 5H),7.64 (d, J=7.2 Hz, 2H), 7.57 (d, J=8.4 Hz, 2H), 7.47 (t, J=7.2 Hz, 2H),7.42 (d, J=4.0 Hz, 1H), 3.72 (d, J=4.0 Hz, 1H).

4-[2-{4-(4′-chlorophenyl)phenylsulfonamido)}acetamido]benzoic acid (31).The synthesis of 31 achieved in a similar manner to 32. ¹H NMR (CD₃OD,400 MHz) δ 7.95 (d, J=8.4 Hz, 2H), 7.88 (d, J=9.2 Hz, 2H), 7.72 (d,J=8.0 Hz, 2H), 7.53-7.50 (m, 4H), 7.43 (d, J=8.0 Hz, 2H), 3.80 (s, 2H).

Compounds 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 were synthesizedusing the procedure outlined for compound 32 from appropriate acylchlorides and amines.

2-Hydroxy-4-(2-(4-biphenylsulfonyloxy)acetamido)benzoic acid (21). ¹HNMR (CD₃OD, 400 MHz) δ 7.93 (d, J=8 Hz, 2H), 7.73 (d, J=8.4 Hz, 2H),7.18 (d, J=8.4 Hz, 1H), 7.55 (d, J=7.6 Hz, 2H), 7.44-7.35 (3H, m), 7.16(appd, 1H), 6.88 (dd, J=8.4, 1.2 Hz, 1H), 3.77 (s, 2H).

4-(2-(Benzyloxy)acetamido)benzoic acid (22). ¹H NMR (CD₃OD, 400 MHz) δ7.97 (d, J=6.4 Hz, 2H), 7.71 (2H, J=6.8 Hz, 2H), 7.44-7.31 (m, 5H), 4.68(s, 2H), 4.11 (s, 2H).

4-(2-(Benzyloxy)acetamido)-2-hydroxybenzoic acid (23). ¹H NMR (CD₃OD,400 MHz) δ 7.78 (d, J=8.0 Hz, 1H), 7.42-7.30 (m, 6H), 7.07-7.05 (d,J=8.0 Hz, 1H), 4.66 (s, 2H), 4.08 (s, 2H).

4-(3-(Phenylsulfonyl)propanamido)-2-hydroxybenzoic acid (24). ¹H NMR(CD₃OD, 400 MHz) δ 7.95-7.91 (m, 4H), 7.70-7.56 (m, 5H), 3.60 (t, J=8.0Hz, 2H), 2.81 (t, J=8.0 Hz).

4-(3-(Phenylsulfonyl)propanamido)benzoic acid (25). ¹H NMR (CD₃OD, 400MHz) δ 7.94 (d, J=8.0 Hz, 4H), 7.73 (d, J=8.8 Hz, 4H), 7.69 (d, J=7.6Hz, 2H), 7.63-7.59 (m, 2H), 7.20 (d, J=2.0 Hz, 1H), 6.93 (dd, J=8.4, 1.6Hz, 1H), 3.60 (t, J=7.2 Hz, 2H), 2.79 (t, J=6.8 Hz, 2H).

4-(4-Phenylbutanamido)-2-hydroxybenzoic acid (26). ¹H NMR (CD₃OD, 400MHz) δ 7.31 (appd, J=1.6 Hz, 1H), 7.27-7.13 (m, 5H), 7.01 (dd, J=8.4,1.6 Hz, 1H), 2.67 (t, J=7.2 Hz, 2H), 2.38 (t, J=6.8 Hz, 2H), 2.02-1.95(m, 2H).

4-(4-Phenylbutanamido)benzoic acid (27). ¹H NMR (CD₃OD, 400 MHz) δ 7.95(d, J=8.8 Hz, 2H), 7.66 (d, J=8.8 Hz, 2H), 7.27-7.15 (m, 5H), 2.68 (t,J=8 Hz, 2H), 2.40 (t, J=8.0 Hz, 2H), 2.00 (m, 2H).

2-(Biphenyl-4-ylsulfonamido)-N-(4-cyanophenyl)acetamide (28). ¹H NMR(CD₃OD, 400 MHz) δ 8.09 (broad t, 1H), 8.02 (d, J=8.0 Hz, 1H), 7.87 (d,J=8.8 Hz, 2H), 7.75-7.64 (m, 5H), 7.47-7.43 (m, 4H), 4.53 (s, 1H), 3.96(appd, 1H).

4-(2-(Biphenyl-4-ylsulfonamido)acetamido)benzenesulfonic acid (29). ¹HNMR (CD₃OD, 400 MHz) δ 7.94 (d, J=8.4 Hz, 2H), 7.75 (d, J=8.0 Hz, 2H),7.71 (d, J=8.0 Hz, 2H), 7.58 (d, J=8.0 Hz, 2H), 7.50-7.38 (m, 6H), 3.78(s, 2H).

2-(Biphenyl-4-ylsulfonamido)-N-(4-sulfamoylphenyl)acetamide (30). ¹H NMR(CD₃OD, 400 MHz) δ 10.27 (s, 1H), 8.16 (broad t, 1H), 7.84 (m, 4H),7.69-7.63 (m, 6H), 7.48 (broad t, 2H), 7.43 (d, J=6 Hz, 1H), 7.23 (s,2H), 3.72 (appd, J=6 Hz, 2H).

The following compounds were prepared according to Scheme 4

4-(tert-Butoxycarbonylamino)-3-(4-chlorophenyl)butanoic acid (33). RefJ. of Peptide Research, 2003, 61, 331. BOC₂O (5.32 g, 24.41 mmol, 1 eq)was added to a stirred solution of Baclofen (5.20 g, 24.41 mmol) andNaOH 1M (73 ml) in water (73 ml) and 1,4-dioxane (73 ml) at 0° C. Afterstirring for 4 h at room temperature, a 10% aqueous solution o citricacid was added until pH 3. The formed white solid was filtered, washedwith water (50 ml) and dried. The pure acid 33 was obtained withoutfurther purification (6.43 g, 20.56 mmol, 84%), mp 139-141° C. ¹H NMR(400 MHz, CD₃OD) δ 1.37 (9H, s), 2.50 (1H, dd, J 8.8, 15.2 Hz), 2.66(1H, dd, J 5.0, 15.4 Hz), 3.12-3.27 (3H, m), 7.22 (2H, d, J 8.4 Hz),7.27 (2H, d, J 8.4 Hz).

tert-Butyl 2-(4-chlorophenyl)-4-oxo-4-(phenylamino)butylcarbamate (34).To a solution of 33 (0.206 g, 0.657 mmol, 1.2 eq) and aniline (0.051 g,0.548 mmol) in dry DMF (5 ml) HATU (0.315 g, 0.9087 mmol, 1.5 eq) andDIPEA (0.340 g, 2.63 mmol, 4 eq) was added at room temperature under Ar.After stirring overnight at room temperature, the reaction mixture wasquenched with water (10 mL) and extracted with ethyl acetate (2×15 ml).The organic extracts were collected, dried over Na₂SO₄ and the solventwas distilled off under reduced pressure. The crude compound waspurified by chromatography on silica gel (3:7 ethyl acetate/hexane, v/v,R_(f) 0.30) to give 34 as a white solid (0.120 g, 0.307 mol, 47%), mp165-167° C. ¹H NMR (400 MHz, CDCl₃) δ 1.43 (9H, s), 2.55 (1H, dd, J 5.0,13.8 Hz), 2.75 (1H, dd, J 8.4, 13.6 Hz), 3.16-3.39 (2H, m), 3.52-3.57(1H, m), 4.60 (1H, bs), 7.07-7.13 (3H, m), 7.29-7.32 (4H, m), 7.54 (2H,d, J 8.4 Hz), 8.61 (1H, bs).

Ethyl4-(4-(tert-butoxycarbonylamino)-3-(4-chlorophenyl)butanamido)benzoate(35)

The compound was prepared from 33 and ethyl 4-aminobenzoate in a similarmanner as described for preparation of 34. Chromatography on silica gel(7:3 hexanes/ethyl (R_(f) 0.20) afforded 35 as a brown solid in 72%yield. ¹H NMR (400 MHz, CD₃OD) δ 1.35 (9H, s, C(CH ₃)₃), 1.35 (3H, t, J6.8 Hz, CH₂CH ₃), 2.63 (1H, dd, J 8.6, 14.6 Hz, H—CHCO), 2.77 (1H, dd, J6.2, 14.6 Hz, H—CHCO), 3.35-3.40 (1H, m, CH₂CHCH₂), 4.31 (2H, q, J 7.2Hz, CH ₂CH₃), 7.24 (2H, d, J 8.8 Hz, 2×Ar—H), 7.28 (2H, d, J 8.8 Hz,2×Ar—H), 7.57 (2H, d, J 8.4 Hz, 2×Ar—H), 7.91 (2H, d, J 9.2 Hz, 2×Ar—H).¹³C NMR (400 MHz, CDCl₃) δ 14.56 (s) (CH₂ CH₃), 28.56 (s) (C(CH₃)₃),40.92 (s) (CH₂CO), 42.77 (s) (CH₂ CHCH₂), 45.97 (s) (CH₂NH), 61.04 (s)(C(CH₃)₃), 80.56 (s) (CH₂CH₃), 119.07 (s) (2×CH, Ar), 125.90 (s) (C,Ar), 129.08 (s) (2×CH, Ar), 129.17 (s) (2×CH, Ar), 130.90 (s) (2×CH,Ar), 133.17 (s) (C, Ar), 140.35 (s) (C, Ar), 142.65 (s) (C, Ar), 157.54(s) (C═O), 166.47 (s) (C═O), 170.32 (s) (C═O). ν_(max) (puresample)/(cm⁻¹)), 3353 (md), 1685 (st) (C═O), 1685 (st) (C═O), 1661 (st)(C═O), 1524 (st), 1273 (st), 1249 (st), 1188 (st), 769 (md).

4-Phenoxybenzenesulfonyl Chloride.

A solution of 4-phenoxybenzenesulfonic acid (3.14 g, 12.64 mmol) inthionyl chloride (9 ml) was refluxed for 4 h in presence of DMF (2drops). After cooling to room temperature, diethyl ether (20 ml) wasadded and the resulting white solid was separated by filtration. Thefiltered solution was evaporated in vacuo to give4-phenoxybenzenesulfonyl chloride as a brown oil which was used in thenext step without further purification.

3-(4-Chlorophenyl)-4-(4-methylphenylsulfonamido)-N-phenylbutanamide(36). To a solution of 34 (0.106 g, 0.271 mmol) in DCM (2 ml) TFA (2 ml)was added. After stirring for 2 h at room temperature, the solvent wasdistilled under reduced pressure to give a brown solid. The cruderesidue was dissolved as obtained in 1,4-dioxane:H₂O 1:1 (5 ml) andK₂CO₃ (0.225 g, 1.63 mmol, 6 eq) was added followed by tosyl chloride(0.056 g, 0.298 mmol, 1.1 eq) at room temperature. After stirring for 2h at room temperature, the resulting mixture was evaporated in vacuo todryness. Water (10 ml) was added and the resulting white solid wasseparated by filtration and dried. Pure 36 was obtained without furtherpurification (0.079 g, 0.179 mmol, 66%), mp 200-202° C. ¹H NMR (400 MHz,CD₃OD) δ 2.39 (3H, s, Ar—CH ₃), 2.56 (1H, dd, J 9.2, 14.4 Hz, H—CHCO),2.77 (1H, dd, J 6.8, 14.4 Hz, H—CHCO), 3.06 (1H, dd, J 8.2, 13.0 Hz,H—CHNH), 3.15 (1H, dd, J 6.8, 13.2 Hz, H—CHNH) 7.03-7.07 (1H, m, CH,Ar), 7.15 (2H, d, J 8.4 Hz, 2×CH, Ar), 7.21-7.26 (4H, s, 4×CH, Ar), 7.29(2H, d, J 8.0 Hz, 2×CH, Ar), 7.36-7.38 (2H, m, 2×CH, Ar), 7.62 (2H, d, J8.8 Hz, 2×CH, Ar). ¹³C NMR (400 MHz, DMSO-d₆) δ 21.63 (s) (Ar—CH₃),40.73 (s) (CH₂CO), 41.87 (s) (CH₂ CHCH₂), 48.13 (s) (CH₂NH), 119.79 (s)(2×CH, Ar), 123.80 (s) (CH, Ar), 127.14 (s) (2×CH, Ar), 128.80 (s)(2×CH, Ar), 129.29 (s) (2×CH, Ar), 130.23 (s) (2×CH, Ar), 130.36 (s)(2×CH, Ar), 131.80 (s) (C, Ar), 138.14 (s) (C, Ar), 139.64 (s) (C, Ar),141.53 (s) (C, Ar), 143.20 (s) (C, Ar), 169.81 (s) (C═O). ν_(max) (puresample)/(cm⁻¹) 3340 (md) (N—H), 3141 (md) (N—H), 1665 (st) (C═O), 1598(st), 1547 (st), 1493 (md), 1444 (st), 1154 (st), 1087 (md). MS(API-ES): m/z 443 [M+H]⁺ (100%), HRMS (API-ES) m/z found 443.1201[M+H]⁺.

Ethyl4-(4-(4-benzylphenylsulfonamido)-3-(4-chlorophenyl)butanamido)benzoate(37). The compound was obtained as a white solid in 65% from 35 and4-phenoxybenzenesulfonyl chloride in a similar manner as described forpreparation of 36, mp. ¹H NMR (400 MHz, CD₃OD) δ 1.36 (3H, t, J 6.8 Hz,CH₂CH ₃), 2.60 (1H, dd, J 8.0, 14.8 Hz, H—CHCO), 2.82 (1H, dd, J 6.0,14.8 Hz, H—CHCO), 3.09 (1H, dd, J 7.8, 13.4 Hz, H—CHNH), 3.16 (1H, dd, J6.4, 13.2 Hz, H—CHNH), 4.32 (2H, q, J 7.2 Hz, CH ₂CH₃), 6.99 (2H, d, J7.2 Hz), 7.06-7.08 (2H, m), 7.18 (2H, d, J 8.4 Hz), 7.18 (2H, d, J 8.4Hz), 7.19-7.24 (3H, m), 7.40-744 (2H, m), 7.56 (2H, d, J 8.8 Hz), 7.72(2H, d, J 8.8 Hz), 7.91 (2H, d, J 8.8 Hz). MS (API-ES): m/z 593 [M+H]⁺(100%), HRMS (API-ES) m/z found 593.15134 [M+H]⁺.

Ethyl4-(3-(4-chlorophenyl)-4-(4-methylphenylsulfonamido)butanamido)benzoate(38). The compound was obtained as a white solid in 96% from 35 andtosyl chloride in a similar manner as described for preparation of 28,mp 187-189° C. ¹H NMR (400 MHz, CD₃OD) δ 1.36 (3H, t, J 7.2 Hz, CH₂CH₃), 2.39 (3H, s, Ar—CH ₃), 2.60 (1H, dd, J 8.8, 14.8 Hz, H—CHCO), 2.82(1H, dd, J 6.0, 14.8 Hz, H—CHCO), 3.06 (1H, dd, J 8.0, 13.2 Hz, H—CHNH),3.14 (1H, dd, J 6.8, 13.2 Hz, H—CHNH) 4.32 (2H, q, J 7.2 Hz, CH ₂CH₃),7.15 (2H, d, J 8.8 Hz), 7.22 (2H, d, J 8.4 Hz), 7.29 (2H, d, J 8.4 Hz),7.54 (2H, d, J 8.8 Hz), 7.62 (2H, d, J 8.4 Hz), 7.91 (2H, d, J 9.2 Hz).¹³C NMR (400 MHz, DMSO-d₆) δ 14.88 (s) (CH₂ CH₃), 21.62 (s) (Ar—CH₃),40.74 (s) (CH₂CO), 41.76 (s) (CH₂ CHCH₂), 48.25 (s) (CH₂NH), 61.08 (s)(CH₂CH₃), 119.06 (s) (2×CH, Ar), 124.78 (s) (C, Ar), 127.34 (s) (2×CH,Ar), 128.83 (s) (2×CH, Ar), 130.23 (s) (2×CH, Ar), 130.36 (s) (2×CH,Ar), 130.84 (s) (2×CH, Ar), 131.83 (s) (C, Ar), 138.15 (s) (C, Ar),141.42 (s) (C, Ar), 143.20 (s) (C, Ar), 143.95 (s) (C, Ar), 165.98 (s)(C═O), 170.47 (s) (C═O). ν_(max) (pure sample)/(cm⁻¹) 3320 (md), 3292(md), 1712 (st) (C═O), 1677 (st) (C═O), 1598 (md), 1540 (st), 1315 (st),1272 (st), 1149 (st), 1104 (st). MS (API-ES): m/z 515 [M+H]⁺ (100%),HRMS (API-ES) m/z found 515.1411 [M+H]⁺.

4-(3-(4-Chlorophenyl)-4-(4-methylphenylsulfonamido)butanamido)benzoicacid (39). A solution of 36 (0.023 g, 0.044 mmol) in methanol (1 ml) andTHF (1 ml) was stirred in presence of NaOH 1M (1 ml) overnight at rt.The solution remaining was concentrated in vacuo. HCl 1M (1 ml) wasadded and the formed white solid was filtered, washed with water (5 ml)and dried. Pure 39 was obtained without further need for purification(0.015 g, 0.030 mmol, 68%), mp 250-252° C. ¹H NMR (400 MHz, CD₃OD) δ2.39 (3H, s, Ar—CH ₃9, 2.60 (1H, dd, J 8.8, 14.8 Hz, H—CHCO), 2.82 (1H,dd, J 6.4, 14.8 Hz, H—CHCO), 3.06 (1H, dd, J 8.0, 13.2 Hz, H—CHNH), 3.14(1H, dd, J 6.8, 13.2 Hz, H—CHNH), 7.16 (2H, d, J 8.8 Hz), 7.23 (2H, d, J8.4 Hz), 7.29 (2H, d, J 8.0 Hz), 7.54 (2H, d, J 9.2 Hz), 7.62 (2H, d, J8.4 Hz), 7.92 (2H, d, J 8.8 Hz). ¹³C NMR (100 MHz, DMSO-d₆) δ. ν_(max)(pure sample)/(cm⁻¹) 1864 (st), 1665 (st), 1653 (st), 1604 (st), 1519(st), 1455 (st), 1324 (st), 1290 (st), 1164 (st), 1151 (st). MS(API-ES): m/z 485 [M−H]⁻ (100%), HRMS (API-ES) m/z found 485.0935 [M−H].

The following compounds were prepared according to Scheme 5.

4-(3-(4-Chlorophenyl)-4-(4-phenoxyphenylsulfonamido)butanamido)benzoicacid (40). A solution of ester 38 (0.065 g, 0.107 mmol) in methanol (1.5ml) and THF (1.5 ml) was stirred in presence of NaOH 1M (1 ml) for 2 hat 80° C. After cooling to rt, the solution remaining was concentratedin vacuo. HCl 1M (1.5 ml) was added and the formed white solid wasfiltered, washed with water (10 ml) and dried. The pure acid 40 wasobtained without further need for purification (0.050 g, 0.088 mmol,83%), mp 119-121° C. ¹H NMR (400 MHz, CD₃OD) δ 2.61 (1H, dd, J 8.4, 14.8Hz, H—CHCO), 2.82 (1H, dd, J 6.0, 14.8 Hz, H—CHCO), 3.09 (1H, dd, J 7.8,13.4 Hz, H—CHNH), 3.16 (1H, dd, J 6.4, 13.2 Hz, H—CHNH), 6.99 (2H, d, J9.2 Hz), 7.06-7.08 (2H, m), 7.17-7.24 (5H, m), 7.47.44 (2H, m), 7.54(2H, d, J 9.2 Hz), 7.72 (2H, d, J 8.8 Hz), 7.91 (2H, d, J 9.2 Hz). ¹³CNMR (400 MHz, CD₃OD) δ. ν_(max) (pure sample)/(cm⁻¹) 3359 (st) (OH),1686 (st) (C═O), 1595 (st) (C═O), 1530 (st), 1487 (st), 1410 (md), 1318(md), 1246 (st), 1150 (st) 1092 (md). MS (API-ES): m/z 563 [M+H]⁺(100%), HRMS (API-ES) m/z found 563.10436 [M−H]⁻.

Ethyl 4-(3-naphthalen-2-yl-acryloylamino)benzoate (42). Anhydrouspyridine (0.331 g, 4.2 mmol, 1.2 eq) and ethyl 4-aminobenzoate (0.577 g,3.50 mmol) were added to a solution of (E)-3-(2-naphthyl)propenoylchloride (prepared from its corresponding acid) (0.757 g, 3.50 mmo) inanhydrous DCM (15 ml) under Ar. After stirring overnight at roomtemperature, the white precipitate was filtered, washed with DCM (10 ml)and dried under vacuum to give the amide 42 (0.960 g, 2.78 mmol, 80%) asa white solid, mp 173-175° C. ¹H NMR (400 MHz, DMSO-d₆) δ 1.30 (3H, t, J7.2 Hz), 4.28 (2H, t, J 7.2 Hz), 6.97 (1H, d, J 15.4 Hz), 7.54-7.57 (2H,m), 7.7 (1H, d, J 15.4 Hz), 7.77 (1H, dd, J 1.6, 9.2 Hz), 7.84 (2H, d, J8.8 Hz), 7.93-7.97 (5H, m), 8.15 (1H, s), 10.58 (1H, bs). ¹³C NMR (100MHz, DMSO-d₆) δ 14.88, 61.09, 119.30, 122.91, 124.18, 124.99, 127.47,127.83, 128.38, 129.09, 129.36, 130.05, 130.98, 132.82, 133.68, 134.26,141.64, 144.33, 164.69, 166.01. ν_(max)(solid)/(cm⁻¹) 3357, 1702, 1660,1619, 1607, 1591, 1521, 1403, 1282. MS (API-ES): m/z 346 [M+H]⁺ (100%),HRMS (API-ES) m/z found 346.1443 [M+H]⁺.

Methyl 4-(3-phenylacryloylamino)benzoate (41). This was obtained as awhite solid from cinnamoyl chloride (1.1 g, 6.62 mmol) and ethyl4-aminobenzoate (1.10 g, 6.62 mmol) in a similar manner as described forpreparation of 42. The reaction mixture was stirred at room temperatureovernight and the mixture was poured in HCl (aq, 2N, 20 ml). The productwas extracted with DCM (2×20 ml), dried over Na₂SO₄ and the solventremoved under reduced pressure. The crude amide 41 was used in the nextstep without further purification.

Methyl 2-hydroxy-4-(3-naphthalen-2-yl-acryloylamino)benzoate (43). Thiswas obtained from (E)-3-(2-naphthyl)propenoyl chloride (0.320 g, 1.48mmol) and methyl 4-amino-2-hydroxybenzoate (0.247 g, 1.48 mmol) in asimilar manner as described for preparation of 42. After stirringovernight at room temperature, the white solid was filtered, washed withDCM (ml) and dried under vacuum to give the amide 43 (0.400 g, 1.15mmol, 79%) as an off-white solid, mp 210-212° C. ¹H NMR (400 MHz,DMSO-d₆) δ 3.86 (3H, s, OCH₃), 6.31 (1H, d, J 15.6 Hz, CH), 7.17 (1H,dd, J 1.8, 8.8 Hz, H-6′), 7.53 (1H, d, J 1.8 Hz, H-2′), 7.54-7.57 (2H,m, 2×CH, Ar), 7.74-7.79 (3H, m, 3×CH, Ar), 7.92-7.99 (3H, m, 3×CH, Ar),8.15 (1H, s, CH, Ar), 10.54 (1H, s, NH), 10.65 (1H, s, NH). ¹³C NMR (100MHz, DMSO-d₆) δ 52.01 (s) (OCH₃), 106.95 (s) (CH, Ar), 108.27 (s) (C,Ar), 111.30 (s) (CH, Ar), 122.78 (s) (CH), 124.21 (s) (CH, Ar), 127.54(s) (CH, Ar), 127.92 (s) (CH, Ar), 128.40 (s) (CH, Ar), 129.12 (s) (CH,Ar), 129.39 (s) (CH, Ar), 130.12 (s) (CH, Ar), 131.56 (s) (CH, Ar),132.76 (s) (C, Ar), 133.67 (s) (C, Ar), 134.30 (s) (C, Ar), 141.91 (s)(CH), 146.35 (s) (C, Ar), 161.91 (s) (C, Ar), 164.84 (s) (C═O), 169.70(s) (C═O). ν_(max) (solid)/(cm⁻¹) 3352 (st), 1692 (st), 1662 (st), 1622(st), 1597 (st), 1507 (st), 1445 (st), 1362 (st), 1265 (st), 1188 (st),1143 (st), 1095 (st). MS (API-ES): m/z 346 [M−H]⁻ (100%), HRMS (API-ES)m/z found 346.1088 [M−H]⁻.

Methyl 4-[3-(4-chlorophenyl)acryloylamino]-2-hydroxy-benzoate (45). Thiswas obtained from 4-chlorocinnamoyl chloride (1.80 g, 9.47 mmol) andmethyl 4-amino-2-hydroxybenzoate (1.58 g, 9.47 mmol) in a similar manneras described for preparation of 42. After stirring for 2 h at roomtemperature, the white precipitate was filtered, washed with DCM (10 ml)and dried under vacuum to give the amide 45 (2.41 g, 7.50 mmol, 80%) asan off-white solid, mp 208-210° C. ¹H NMR (400 MHz, DMSO-d₆) 3.85 (3H,s, OCH₃), 6.80 (1H, d, J 15.8 Hz, CH), 7.13 (1H, dd, J 2.4, 8.8 Hz,H-6′), 7.49 (1H, d, J 2.4 Hz, H-2′), 7.50 (2H, d, J 8.4 Hz, 2×CH, Ar),7.60 (1H, d, J 15.8 Hz, CH), 7.65 (2H, s, J 8.4 Hz, 2×CH, Ar), 7.74 (1H,s, J 8.8 Hz, H-5′), 10.50 (1H, s, NH), 10.63 (1H, s, OH). ¹³C NMR (100MHz, DMSO-d₆) δ 52.89 (s) (OCH₃), 106.97 (s) (CH, Ar), 108.32 (s) (C,Ar), 111.28 (s) (CH, Ar), 123.20 (s) (CH), 129.75 (s) (2×CH, Ar), 130.20(s) (2×CH, Ar), 131.47 (s) (CH, Ar), 134.15 (s) (C, Ar), 135.14 (s) (C,Ar), 140.47 (s) (C, Ar), 146.24 (s) (CH), 161.92 (s) (C, Ar), 164.58 (s)(C═O), 169.70 (s) (C═O). ν_(max)(solid)/(cm⁻¹) 3346 (st), 1693 (st),1660 (st), 1628 (st), 1597 (st), 1507 (st), 1442 (st), 1269 (st), 1189(st). MS (API-ES): m/z 330 [M³⁵Cl—H]⁻ (100%), 332 [M³⁷Cl—H]332 (35%).HRMS (API-ES) m/z found 330.0552 [M−H]⁻.

Ethyl 4-(4-nitro-3-phenyl-butyrylamino)benzoate (47). A mixture of 41(1.340 g, 4.44 mmol) in nitromethane (4 ml) was heated a microwavereactor (Biotage Initiator) at 100° C. for 15 min in presence of DBU(0.811 g, 5.328 mmol, 1.2 eq). After cooling to room temperature, thereaction mixture was poured into HCl 1M (10 ml). The product extractedwith ethyl acetate (2×15 ml). The organic extracts were collected, driedover Na₂SO₄ and the solvent removed under reduced pressure.Chromatography on silica gel (7:3 hexanes/ethyl acetate, R_(f) 0.20)afforded 47 (0.980 g, 2.75 mmol, 62%) as a yellow oil. ¹H NMR (400 MHz,CDCl₃) δ 1.37 (3H, t, J 7.6 Hz), 2.79 (1H, dd, J 7.2, 15.2 Hz), 2.86(1H, dd, J 7.2, 15.2 Hz), 4.07 (1H, quint, J 7.2 Hz), 4.34 (2H, q, J 7.2Hz), 4.72 (1H, dd, J=7.8, 12.6 Hz), 4.83 (1H, dd, J 6.4, 12.4 Hz), 6.63(1H, d, J 16 Hz), 7.11 (1H, t, J 7.6 Hz), 7.24-7.26 (2H, m), 7.27-7.36(2H, m), 7.45 (1H, s), 7.47 (2H, d, J 8.8 Hz), 7.96 (2H, d, J 8.8 Hz).¹³C NMR (400 MHz, CDCl₃) δ 14.55, 40.67, 40.93, 61.20, 79.50, 119.17,126.54, 127.53, 128.45, 129.48, 130.98, 138.51, 141.60, 166.30, 168.39.MS (API-ES): m/z found 357 [M+H]⁺ (100%), HRMS (API-ES) m/z found357.1344 [M+H]⁺.

Ethyl 4-(3-naphthalen-2-yl-4-nitro-butyrylamino)benzoate (48). This wasprepared from corresponding amide 42 (0.995 g, 2.88 mmol) in a similarmanner as described for preparation of 47. Chromatography on silica gelperformed using a FlashMaster 3 purification station (75:25hexanes/ethyl acetate, R_(f) 0.20) afforded 48 (0.819 g, 2.017 mmol,70%) as a yellow oil. ¹H NMR (400 MHz, DMSO-d₆) δ 1.26 (3H, t, J 7.6Hz), 2.85-2.93 (2H, m), 4.10 (1H, quint, J 8.0 Hz), 4.23 (1H, q, J 7.2Hz), 5.02-5.10 (2H, m), 7.45-7.48 (2H, m), 7.54 (1H, dd, J 1.6, 8.8 Hz),7.62 (2H, d, J 8.8 Hz), 7.82-7.87 (6H, m). ¹³C NMR (400 MHz, CDCl₃) δ14.52, 40.75, 40.79, 61.21, 79.48, 119.21, 124.97, 126.43, 126.65,126.78, 126.87, 127.92, 128.04, 129.37, 130.94, 133.11, 133.59, 135.89,141.68, 166.37, 168.54. MS (API-ES): m/z found 407 [M+H]⁺ (100%), HRMS(API-ES) m/z 407.1607 [M+H]⁺.

Methyl 2-(benzyloxy)-4-(3-(naphthalen-2-yl)-4-nitrobutanamido)benzoate(49). Benzyl bromide (0.236 g, 1.38 mmol) and K₂CO₃ (0.190 g, 1.38 mmol)were added to a stirred solution of 43 (0.400 g, 1.15 mmol) in anhydrousDMF (7 ml) under Ar. The reaction mixture was stirred at roomtemperature overnight and the mixture was poured in water (15 ml). Theproduct was extracted with ethyl acetate (2×15 ml), dried over Na₂SO₄and the solvent removed under reduced pressure. The resultant product 44was stirred in the microwave reactor in presence of DBU (0.192 g, 1.27mmol), and nitromethane (7 ml) in the microwave), at 100° C. for 15 min.After cooling to room temperature, the reaction mixture was poured intoHCl 1M (10 ml). The product was extracted with ethyl acetate (2×15 ml),dried over Na₂SO₄ and the solvent removed under reduced pressure.Chromatography on silica gel (60:40 hexanes/ethyl acetate) afforded 49(0.413 g, 0.83 mmol, 72%) as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ2.88 (1H, dd, J 6.8, 15.6 Hz), 2.96 (1H, dd, J 7.2, 15.2 Hz), 3.86 (3H,s), 4.26 (1H, quint, J 7.1 Hz), 4.83 (1H, dd, J 7.4, 12.6 Hz), 4.93 (1H,dd, J 6.8, 12.4 Hz), 5.10 (2H, s), 6.71 (1H, dd, J 2.0, 8.8 Hz), 7.13(1H, bs), 7.28-7.32 (1H, m), 7.36-7.39 (3H, m), 7.47-7.52 (5H, m), 7.71(1H, bs), 7.77-7.86 (4H, m). ¹³C NMR (400 MHz, CDCl₃) δ 40.68, 40.81,52.15, 70.70, 79.44, 101.39, 111.15, 115.91, 124.94, 126.67, 126.79,126.89, 127.22, 127.93, 128.05, 128.79, 128.72, 129.39, 133.11, 133.60,135.89, 136.54, 142.68, 159.61, 166.42, 168.57. MS (API-ES): m/z found499 [M+H]⁺ (100%), HRMS (API-ES) m/z 499.1862 [M+H]⁺.

Methyl 2-(benzyloxy)-4-(3-(4-chlorophenyl)-4-nitrobutanamido)benzoate(50). Benzyl bromide (0.575 g, 3.36 mmol) and K₂CO₃ (0.77 g, 5.60 mmol)were added to a stirred solution of 45 (0.9 g, 2.80 mmol) in anhydrousDMF (20 ml) under Ar. The reaction mixture was stirred at roomtemperature overnight and the mixture was poured in water (20 ml). Theproduct was extracted with ethyl acetate (2×20 ml), dried over Na₂SO₄and the solvent removed under reduced pressure. The resultant crude 46was stirred in the microwave reactor in presence of DBU (0.468 g, 3.08mmol), and nitromethane (10 mL in the microwave), at 100° C. for 15 min.After cooling to room temperature, the reaction mixture was poured intoHCl 1M (15 ml). The product was extracted with ethyl acetate (2×20 ml),dried over Na₂SO₄ and the solvent removed under reduced pressure.Chromatography on silica gel (80:20, hexanes/ethyl acetate) afforded 50(0.521 g, 1.10 mmol, 40%) as a white solid, mp 81-83° C. ¹H NMR (400MHz, CDCl₃) δ 2.75 (1H, dd, J 6.6, 15.4 Hz), 2.85 (1H, dd, J 7.6, 15.6Hz), 3.87 (3H, s), 4.07 (1H, quint, J 6.8 Hz), 4.70 (1H, dd, J 7.8, 13.0Hz), 4.83 (1H, dd, J 6.4, 12.8 Hz), 5.17 (2H, s), 6.77 (1H, dd, J 2.0,8.4 Hz), 7.19 (3H, d, J 8.0 Hz), 7.32 (3H, d, J 8.8 Hz), 7.39 (2H, t, J8.0 Hz), 7.51 (2H, d, J 7.2 Hz), 7.55 (1H, bs), 7.82 (1H, d, J 8.0 Hz).¹³C NMR (100 MHz, CDCl₃) δ 39.89, 40.59, 52.18, 70.79, 79.25, 104.96,111.08, 116.14, 127.20, 128.11, 128.76, 128.92, 129.61, 133.17, 134.28,136.56, 137.04, 142.50, 159.65, 166.36, 168.17. ν_(max) (puresample)/(cm⁻¹) 3329, 1695, 1591, 1549, 1530, 1245, 1090. MS (API-ES):m/z 483 [M+H]⁺ (100%), HRMS (API-ES) m/z found 483.1236 [M+H]⁺.

Ethyl 4-(4-amino-3-phenylbutanamido)benzoate (51). Ref. J. Org. Chem.,2000, 65, 8001. To a stirred solution of nitro compound 47 (0.749 g,2.10 mmol) and NiCl₂.6H₂O (1.99 g, 8.40 mmol) in methanol (10 ml) NaBH₄(0.719 g, 18.93 mmol) was added portionwise over 20 min at 0° C. Afterstirring for 15 at room temperature, the solvent was removed underreduced pressure. Water (20 ml) and ethyl acetate (40 ml) were added tothe solid residue. The resulting mixture was filtered through a celitebed which was washed with ethyl acetate (20 ml). After collecting thefiltrate, the organic phase was separated, dried over Na₂SO₄ and thesolvent removed under reduced pressure to afford amine 51 as an offwhite solid (0.596 g, 1.83 mmol, 87%). The amine was used in the nextstep without further purification.

Ethyl 4-(4-amino-3-(naphthalen-2-yl)butanamido)benzoate (52). This wasobtained as a yellow solid (0.819 g, 2.178 mmol, 89%) from nitrocompound 48 (0.995 g, 2.450 mmol) in a similar manner as described forpreparation of 51. The product amine 52 used in the next step withoutfurther purification.

Methyl 4-(4-amino-3-(naphthalen-2-yl)butanamido)-2-(benzyloxy)benzoate(53). This was obtained as a white solid (0.023 g, 0.033 mmol, 36%) fromnitro compound 49 (0.043 g, 0.091 mmol) a similar manner as describedfor preparation of 51. The product amine 53 was taken to the next stepwithout further purification.

Methyl 4-(4-amino-3-(4-chlorophenyl)butanamido)-2-(benzyloxy)benzoate(54). This was obtained as yellow solid (0.349 g, 0.79 mmol, 77%) fromnitro compound 50 (0.488 g, 1.033 mmol) a similar manner as describedfor preparation of 51. The crude amine 54 was taken to the next stepwithout further purification.

Ethyl 4-(4-(4-methylphenylsulfonamido)-3-phenylbutanamido)benzoate (55).Potassium carbonate (0.325 g, 2.35 mmol) was added to a solution of 51(0.128 g, 0.392 mmol) in 1,4-dioxane/H₂O 1:1 (5 mL) and followed bytosyl chloride (0.074 g, 0.392 mmol) at room temperature. After stirringfor 2 h at room temperature, the resulting mixture was evaporated invacuo to dryness. Water (10 ml) was added and the resulting white solidwas separated by filtration and dried in vacuo. Pure 55 was obtained asan off-white solid (0.122 g, 0.254 mmol, 65%) without furtherpurification, mp 135-137° C. ¹H NMR (400 MHz, CD₃OD) 1.36 (3H, t, J 7.2Hz), 2.39 (3H, s), 2.63 (1H, dd, J 8.6, 14.6 Hz), 2.85 (1H, dd, J 6.2,15.0 Hz), 3.05 (1H, dd, J 7.4, 13.0 Hz), 3.12 (1H, dd, J 6.8, 11.8 Hz),4.32 (2H, q, J 7.2 Hz), 7.17-7.19 (3H, m), 7.23-7.27 (2H, m), 7.31 (2H,d, J 8.0 Hz), 7.54 (2H, d, J 8.4 Hz), 7.65 (2H, d, J 8.4 Hz), 7.90 (2H,d, J 8.8 Hz). ¹³C NMR (100 MHz, CDCl₃) 14.57, 21.73, 40.65, 42.04,47.77, 61.07, 119.15, 127.14, 127.75, 127.77, 129.29, 130.07, 130.94,136.79, 140.74, 142.12, 143.97, 166.36, 170.24. ν_(max) (solid)/(cm⁻¹)3324, 3191, 1707, 1675, 1597, 1534, 1270, 1157, 1105. MS (API-ES): m/z481 [M+H]⁺ (100%), HRMS (API-ES) m/z found 481.1801 [M+H]⁺.

Ethyl 4-(4-(4-phenoxyphenylsulfonamido)-3-phenylbutanamido)benzoate(56). This was obtained as an off white solid (0.182 g, 0.326 mmol, 77%)from 51 (0.137 g, 0.420 mmol) and 4-phenoxybenzenesulfonyl chloride(0.112 g, 0.420 mmol) in a similar manner as described for preparationof 55, mp 147-149° C. ¹H NMR (400 MHz, CD₃OD) δ 1.36 (3H, t, J 7.6 Hz),2.63 (1H, dd, J 8.4, 14.8 Hz), 2.84 (1H, dd, J 7.4, 13.2 Hz), 3.09 (1H,dd, J 7.2, 12.8 Hz), 3.16 (1H, dd, J 7.2, 12.4 Hz), 4.31 (2H, q, J 6.8Hz), 7.00 (2H, d, J 7.2 Hz), 7.06 (2H, dd, J 0.8, 8.8 Hz), 7.18-7.22(3H, m), 7.23-7.26 (3H, m), 7.39-741 (2H, m), 7.73 (2H, d, J 8.8 Hz),7.89 (2H, d, J 8.8 Hz). ¹³C NMR (100 MHz, CDCl₃) 14.57, 40.73, 42.06,47.80, 61.07, 117.92, 119.15, 120.52, 125.25, 127.75, 127.80, 129.32,129.35, 130.41, 130.95, 133.27, 140.74, 142.08, 155.23, 161.95, 166.34,170.20. ν_(max) (solid)/(cm⁻¹) 3322, 1702, 1596, 1532, 1487, 1407, 1274,1243, 1151, 1104, 695. MS (API-ES): m/z 559 [M+H]⁺ (100%), HRMS (API-ES)m/z found 559.1906 [M+H]⁺.

Ethyl4-(4-(4-methylphenylsulfonamido)-3-(naphthalen-2-yl)butanamido)benzoate(57). This was obtained as an off white solid (0.058 g, 0.109 mmol) from52 (0.138 g, 0.375 mmol) in a similar manner as described forpreparation of 55. ¹H NMR (400 MHz, CD₃OD) δ 1.34 (3H, t, J 7.2 Hz),2.34 (3H, s), 2.73 (1H, dd, J 8.2, 14.6 Hz), 2.92 (1H, dd, J 6.4, 14.8Hz), 3.17-3.28 (2H, m), 3.47-3.52 (1H, m), 4.30 (2H, q, J 7.2 Hz,CH₂CH₃), 7.19 (2H, d, J 8.0 Hz), 7.32 (1H, dd, J 1.4, 8.6 Hz), 7.40-7.45(2H, m), 7.51 (2H, d, J 8.4 Hz), 7.59 (2H, d, J 8.8 Hz), 7.60 (1H, s),7.73-7.79 (3H, m), 7.90 (2H, d, J 9.2 Hz). ν_(max) (solid)/(cm⁻¹) 2958,1706, 1596, 1531, 1273, 1171, 1152, 1105, 1020. MS (API-ES): m/z 531[M+H]⁺ (100%), HRMS (API-ES) m/z found 531.1962 [M+H]⁺.

Ethyl4-(3-(naphthalen-2-yl)-4-(4-phenoxyphenylsulfonamido)butanamido)benzoate(58). The compound was obtained as a white solid in (0.100 g, 0.164mmol, 73%) from 52 in a similar manner as described for preparation of55, mp 141-143° C. ¹H NMR (400 MHz, CD₃OD) δ 1.34 (3H, t, J 7.2 Hz),2.75 (1H, dd, J 8.4, 14.8 Hz), 2.92 (1H, dd, J 6.6, 15.0 Hz), 3.25-3.29(2H, m), 3.34-3.53 (1H, m) 4.30 (2H, q, J 7.2 Hz), 6.61 (2H, d, J 9.2Hz), 7.05 (2H, dd, J 1.2, 8.8 Hz), 7.20-7.23 (1H, m), 7.34 (1H, dd, J1.4, 8.6 Hz), 7.40-7.45 (4H, m), 7.54 (2H, d, J 8.8 Hz), 6.64 (1H, bs),7.67 (2H, d, J 8.8 Hz), 7.75-7.90 (3H, m), 7.87 (2H, d, J 9.2 Hz). ¹³CNMR (100 MHz, CDCl₃) 14.55, 40.68, 42.12, 47.84, 61.05, 117.89, 119.19,120.51, 125.22, 125.67, 126.21, 126.35, 126.53, 126.73, 127.85, 127.96,129.09, 129.83, 130.39, 130.90, 132.85, 133.27, 133.61, 138.16, 142.13,155.24, 161.90, 166.34, 170.26. ν_(max) (solid)/(cm⁻¹) 1707, 1596, 1531,1486, 1275, 1243, 1151, 1098. MS (API-ES): m/z 609 [M+H]⁺ (100%), HRMS(API-ES) m/z found 609.2058 [M+H]⁺.

4-(4-(4-methylphenylsulfonamido)-3-phenylbutanamido)benzoic acid (59). Asolution of the ester 55 (0.106 g, 0.220 mmol) in methanol (3 ml) andTHF (3 ml) was stirred in presence of NaOH (aq, 1M, 1 ml) at roomtemperature overnight. The solvent was removed under reduced pressure.HCl (aq, 1M, 1.5 ml) was added and the white precipitate was filtered,washed with water (5 ml) and dried under vacuum. Pure 59 was obtained asa white solid (0.085 g, 0.188 mmol, 85%) without further purification,mp 225-227° C. ¹H NMR (400 MHz, CD₃OD) δ 2.39 (3H, s), 2.62 (1H, dd, J8.4, 14.8 Hz), 2.35 (1H, dd, J 6.6, 14.6 Hz), 3.05 (1H, dd, J 8.0, 13.0Hz), 3.12 (1H, dd, J 7.2, 12.8 Hz), 7.16-7.19 (3H, m), 7.24-7.27 (2H,m), 7.30 (2H, d, J 7.6 Hz), 7.53 (2H, d, J 8.4 Hz), 7.65 (2H, d, J 8.4Hz), 7.91 (2H, d, J 9.2 Hz). ¹³C NMR (100 MHz, DMSO-d₃) δ 21.61, 40.65,42.32, 48.33, 118.95, 125.61, 127.18), 127.26, 128.38, 128.96, 130.02,130.98, 138.09, 142.47, 143.22, 143.72, 167.55, 170.64. ν_(max)(solid)/(cm⁻¹) 3316, 3277), 1668, 1595, 1531, 1408, 1317, 1254, 1152. MS(API-ES): m/z 451 [M−H]⁻ (100%), HRMS (API-ES) m/z found 451.1334[M−H]⁻.

4-(4-(4-phenoxyphenylsulfonamido)-3-phenylbutanamido)benzoic acid (60).This was obtained as a white solid (0.120 g, 0.226 mmol, 83%) from theester 56 (0.152, 0.272 mmol) in a similar manner as described forpreparation of 59, mp 187-189° C. ¹H NMR (400 MHz, CD₃OD) δ 2.63 (1H,dd, J 8.8, 14.8 Hz), 2.84 (1H, dd, J 6.6, 14.6 Hz), 3.09 (1H, dd, J 7.8,13.0 Hz), 3.16 (1H, dd, J 6.8, 12.8 Hz), 7.00 (2H, d, J 9.2 Hz), 7.06(2H, dd, J 1.2, 8.4 Hz), 7.16-7.28 (6H, m), 7.39-743 (2H, m), 7.53 (2H,d, J 8.4 Hz), 7.73 (2H, d, J 8.4 Hz), 7.90 (2H, d, J 8.4 Hz). ¹³C NMR(100 MHz, CD₃OD) 40.86, 42.63, 47.85, 117.47, 119.00, 120.09, 124.77,125.74, 126.70, 127.66, 128.46, 129.12, 130.10, 130.49, 134.36, 141.39,142.91, 155.62, 161.59, 168.23, 171.43. ν_(max) (solid)/(cm⁻¹) 3060,1681, 1596, 1585, 1487, 1318, 1294, 1251, 11511. MS (API-ES): m/z found529 [M−H](100%), HRMS (API-ES) m/z found 529.1433 [M−H]⁻.

4-(3-(naphthalen-2-yl)-4-(4-phenoxyphenylsulfonamido)butanamido)benzoicacid (61). This was obtained as a white solid (0.052 g, 0.090 mmol, 70%)from 57 (0.079 g, 0.129 mmol) in a similar manner as described forpreparation of 59, mp 183-184° C. ¹H NMR (400 MHz, CD₃OD) δ 2.65 (1H,dd, J 8.4, 14.8 Hz), 2.82 (1H, dd, J 6.8, 14.8 Hz), 3.11-3.20 (2H, m),3.40-3.44 (1H, m), 6.81 (2H, d, J 8.4 Hz), 6.94-6.96 (2H, m), 7.10-7.14(1H, m), 7.25 (1H, dd, J 1.6, 8.8 Hz), 7.29-7.35 (4H, m), 7.41 (2H, d, J8.8 Hz), 7.54 (1H, bs), 7.58 (2H, d, J 8.8 Hz), 7.65-7.68 (3H, m), 7.78(2H, d, J 8.4 Hz). ¹³C NMR (100 MHz, CD₃OD) 40.82, 42.70, 47.90, 117.34,119.00, 120.115, 124.76, 125.53, 125.66, 125.91, 126.54, 127.39, 127.53,128.12, 129.06, 130.09, 130.51, 132.92, 133.74, 134.38, 138.80, 142.80,155.55, 161.50, 171.36. ν_(max) (solid)/(cm⁻¹) 3061, 1683, 1599, 1584,1514, 1486, 1304, 1240, 1147. MS (API-ES): m/z found 579 [M−H]⁻ (100%),HRMS (API-ES) m/z found 579.1585 [M−H]⁻.

4-(4-(4-methylphenylsulfonamido)-3-(naphthalen-2-yl)butanamido)benzoicacid (62). This was obtained as a white solid (0.021 g, 0.042 mmol, 65%)from 58 (0.034 g, 0.064 mmol) in a similar manner as described forpreparation of 59, mp. ¹H NMR (400 MHz, CD₃OD) δ 2.34 (3H, s), 2.73 (1H,dd, J 8.2, 14.6 Hz), 2.91 (1H, dd, J 6.4, 14.8 Hz), 3.20 (1H, dd, J 7.6,12.8 Hz), 3.25-3.29 (1H, m), 3.47-3.53 (1H, m), 7.19 (2H, d, J 8.0 Hz),7.32 (1H, dd, J 1.6, 8.8 Hz), 7.39-7.45 (2H, m), 7.51 (2H, d, J 9.2 Hz),7.59 (2H, d, J 8.8 Hz), 7.60 (1H, s), 7.73-7.79 (3H, m), 7.88 (2H, d, J9.2 Hz). ¹³C NMR (100 MHz, DMSO-d₆) 21.60, 42.52, 48.31, 118.92, 125.73,126.20, 126.65, 126.88, 126.95, 127.13, 128.09, 128.20, 128.41, 130.18,130.95, 132.73, 133.62, 138.16, 140.03, 143.15, 143.67, 167.57, 170.61.ν_(max) (solid)/(cm⁻¹) 1680, 1595, 1530, 1408, 1305, 1252, 1152. MS(API-ES): m/z 5019 [M−H]⁻ (100%), HRMS (API-ES) m/z found 501.14831[M−H]⁻.

Methyl2-(benzyloxy)-4-(3-(naphthalen-2-yl)-4-(4-phenoxyphenylsulfonamido)butanamido)benzoate(63). This was obtained as an off white solid (0.023 g, 0.032 mmol, 36%)from amine 53 (0.043 g, 0.091 mmol) in a similar manner as described forpreparation of 55. The crude ester was used in the next step withoutfurther purification.

Methyl2-hydroxy-4-(3-(naphthalen-2-yl)-4-(4-phenoxyphenylsulfonamido)butanamido)benzoate(64). A solution of 63 (0.023 g, 0.032 mmol) in methanol (1 ml) wasstirred in presence of ammonium formate (0.089 g) and 10% Pd/C (0.023g), under H₂, at room temperature for 2 days. The solution was thenfiltered through a celite bed and the solvent removed under reducedpressure to afford ester 64 (0.014 g, 0.0230 mmol, 70%) as yellow solid,which was used in the next step without further purification.

2-hydroxy-4-(3-(naphthalen-2-yl)-4-(4-phenoxyphenylsulfonamido)butanamido)benzoicacid (65). This was obtained as an off-white solid (0.0063 g, 0.011mmol, 50%) ester 64 (0.013 g, 0.021 mmol) in a similar manner asdescribed for preparation of 59. mp 153-155° C. ¹H NMR (400 MHz, CD₃OD)δ 2.63 (1H, dd, J 14.6, 8.2 Hz), 2.86 (1H, dd, J 15.0, 6.6 Hz), 3.13(1H, dd, J 13.2, 7.6 Hz), 3.16-3.20 (1H, m), 3.37-3.48 (1H, m),6.77-6.83 (3H, m), 6.94-6.96 (2H, m), 7.08-7.14 (2H, m), 7.24 (1H, dd, J8.4, 1.66 Hz), 7.29-7.35 (4H, m), 7.53 (1H, bs), 7.57-7.60 (1H, m),7.66-7.69 (3H, m), 9.80 (1H, bs). MS (API-ES): m/z 595 [M−H](100%), HRMS(API-ES) m/z found 595.1537 [M−H]⁻.

Methyl2-(benzyloxy)-4-(3-(4-chlorophenyl)-4-(4-phenoxyphenylsulfonamido)butanamido)benzoate(66). This was obtained as an off-white solid (0.205 g, 0.304 mmol, 78%)from the amine 54 (0.137 g, 0.391 mmol) in a similar manner as describedfor preparation of 55. The crude product sulfonamide was taken to thenext step without further purification.

Methyl4-(3-(4-chlorophenyl)-4-(4-phenoxyphenylsulfonamido)butanamido)-2-hydroxybenzoate(67). This was obtained as an off-white solid (0.106 g, 0.181 mmol, 67%)from benzyl ether 66 (0.183 g, 0.27 mmol) in a similar manner asdescribed for preparation of 64. The crude ester 67 was taken to thenext step without further purification.

4-(3-(4-Chlorophenyl)-4-(4-phenoxyphenylsulfonamido)butanamido)-2-hydroxybenzoicacid (68). This was obtained as an off-white solid (0.047 g, 0.082 mmol,67%) from the ester 67 (0.076 g, 0.130 mmol) in a similar manner asdescribed for preparation of 59. The crude acid 68 was taken to the nextstep without further purification. ¹H NMR (400 MHz, CDCl₃) δ 2.59 (1H,dd, J 14.8, 8.8 Hz), 2.80 (1H, dd, J 14.8, 6.8 Hz), 3.12 (2H, m), 6.90(1H, dd, J 10.4, 2.0 Hz), 6.99 (2H, d, J 9.2 Hz), 7.07 (2H, d, J 9.2Hz), 7.16-7.27 (6H, m), 7.40-7.44 (2H, m), 7.69-7.74 (3H, m). MS(API-ES): m/z 579 [M−H]⁻ (100%), HRMS (API-ES) m/z found 579.1000[M−H]⁻.

Ethyl 4-(3-(naphthalen-2-yl)propanamido)benzoate (69). Ref. J. of Org.Chem., 2000, 65, 8001. To a stirred solution of amide 42 (0.086 g, 0.249mmol) and NiCl₂.6H₂O (0.118 g, 0.498 mmol, 2 eq) in methanol (8 ml) andTHF (4 ml) NaBH₄ (0.028 g, 0.749 mmol, 4 eq) was added portionwise over20 min at 0° C. After stirring for 1 h at room temperature, the solventwas distilled off under reduced pressure. Water (10 ml) and ethylacetate (10 ml) was added. The resulting mixture was filtered through acelite bed which was washed with ethyl acetate (20 ml). After collectingthe filtrate, the organic phase was separated, dried over Na₂SO₄ and thesolvent was distilled off under reduced pressure to give the pureproduct 69 as a white solid (0.066 g, 0.190 mmol, 79%), mp 119-121° C.¹H NMR (400 MHz, CDCl₃) δ 1.38 (3H, t, J 7.2 Hz, CH₂CH ₃), 2.75 (2H, t,J 7.2 Hz, CH ₂CO), 3.23 (2H, t, J 7.2 Hz, CH ₂Ar), 4.35 (2H, q, J 7.2Hz, CH ₂CH₃), 7.12 (1H, bs, N—H), 7.37 (1H, dd, J 1.6, 8.4 Hz, CH, Ar),7.42-7.50 (4H, m, 4×CH, Ar), 7.69 (1H, bs, CH, Ar), 7.76-7.82 (3H, m,3×CH, Ar), 7.97 (2H, d, J 9.2 Hz, 2×CH, Ar). MS (API-ES): m/z 348 [M+H]⁺(100%), HRMS (API-ES) m/z found 348.1602 [M+H]⁺.

Ethyl 4-(3-phenylpropanamido)benzoate (70). This was as obtained from 41in a similar manner as described for preparation of 69. The crudeproduct 70 was taken to the next step without further purification, mp109-111° C. ¹H NMR (400 MHz, CDCl₃) δ (3H, t, J Hz, CH₂CH ₃), (2H, t, JHz, CH ₂CO), (2H, t, J Hz, CH ₂,Ar), (2H, q, J 7 Hz, CH ₂CH₃), (1H, bs,N—H), 7.37 (1H, dd, J 1.6, 8.4 Hz, CH, Ar), 7.42-7.50 (4H, m, 4×CH, Ar),7.69 (1H, bs, CH, Ar), 7.76-7.82 (3H, m, 3×CH, Ar), 7.97 (2H, d, J 9.2Hz, 2×CH, Ar). MS (API-ES): m/z 298 [M−H]⁻ (100%), HRMS (API-ES) m/zfound 298.1444 [M−H]⁻.

4-(3-(Naphthalen-2-yl)propanamido)benzoic acid (71). The pure compoundwas obtained as a white solid in 73% yield from 69 in a similar manneras described for preparation of 59, mp 255-257° C. ¹H NMR (400 MHz,CD₃OD) δ 2.69 (2H, t, J 7.2 Hz, CH ₂CO), 3.07 (2H, t, J 7.6 Hz, CH₂—Ar), 7.28-7.35 (3H, m, 3×CH, Ar), 7.53 (2H, d, J 8.4 Hz, 2×CH, Ar),7.61 (1H, bs, CH, Ar), 7.66-7.71 (3H, m, 3×CH, Ar), 7.84 (2H, d, J 9.2Hz, 2×CH, Ar). ¹³C NMR (400 MHz, DMSO-d₆) δ 31.53 (s) (CH₂CO), 38.60 (s)(CH₂Ar), 118.95 (s) (2×CH, Ar), 125.99 (s) (CH, Ar), 126.71 (s) (CH,Ar), 126.80 (s) (CH, Ar), 127.91 (s) (CH, Ar), 127.98 (s) (CH, Ar),128.15 (s) (CH, Ar), 128.46 (s) (CH, Ar), 131.03 (s) (2×CH, Ar), 132.34(s) (C, Ar), 133.82 (s) (C, Ar), 139.41 (s) (C, Ar), 143.85 (s) (C, Ar),167.65 (s) (C═O), 171.62 (s) (C═O). ν_(max) (pure sample)/(cm⁻¹) 3298(st) (OH), 1701 (md) (C═O), 1674 (st) (C═O) 1607 (md), 1595 (md), 1530(st), 1429, 1405, 1293. MS (API-ES): m/z 318 [M−H]⁻ (100%), HRMS(API-ES) m/z found 318.11327 [M−H]⁻.

4-(3-Phenylpropanamido)benzoic acid (72). This was obtained pure as awhite solid in 76% yield from 69 in a similar manner as described forpreparation of 59. ¹H NMR (400 MHz, CD₃OD) δ 2.68 (2H, t, J 7.6 Hz),2.99 (2H, t, J 8.4 Hz), 7.16-7.19 (1H, m), 7.23-7.26 (4H, m), 7.63 (2H,d, J 8.4 Hz), 7.94 (2H, d, J 8.4 Hz). ν_(max) (pure sample)/(cm⁻¹) 3310,1666, 1607 (md), 1593, 1522, 1428, 1404, 1318. MS (API-ES): m/z 268[M−H]⁻ (100%), HRMS (API-ES) m/z 268.0979 [M−H]⁻.

4-(3-Phenylpropanamido)-2-hydroxybenzoic acid (73). A solution of thechlorocinnamide 45 (0.118 g, 0.367 mmol) in methanol (6 ml) and THF (6ml) was stirred in presence of ammonium formate (0.231 g) and 10% Pd/C(0.010 g) at room temperature under Ar. After stirring overnight, thesolution was filtered through a celite bed and the solvent removed underreduced pressure to afford an off-white solid. The solution of the crudecompound in methanol (10 ml) and THF (4 ml) was stirred in presence ofNaOH (aq, 1 M, 3 ml) at 100° C. for 2 h. After cooling to roomtemperature, the solvent was removed under reduced pressure, and theresulting aqueous solution acidified with HCl (aq, 1 M) until pH 1. Theprecipitate was collected by filtration, washed with H₂O (3 ml) anddried in vacuo. Pure 22 was obtained as an off-white solid withoutfurther purification (0.045 g, 0.165 mmol, 45%), mp 165-167° C. ¹H NMR(400 MHz, DMSO-d₆) δ 2.61 (2H, t, J 8.0 Hz), 2.88 (2H, t, J 8.0 Hz),7.00 (1H, dd, J 8.8, 2.4 Hz), 7.21-7.33 (5H, m), 7.67 (2H, d, J 8.8 Hz),MS (API-ES): m/z 284 [M−H]⁻ (100%), HRMS (API-ES) m/z found 284.097194[M−H]⁻.

The following compounds were prepared according to Scheme 6.

6-(Methoxycarbonyl)-2-naphthoic acid (74). A solution of KOH (0.459 g,8.18 mmol) in methanol (1.5 ml) was added to a suspension of dimethyl2,6-naphthlene dicarboxylate (2.0 g, 8.18 mmol) in dioxane (20 ml). Thereaction mixture was stirred at 80° C. for 4 h, cooled to roomtemperature and a solid residue was rinsed with diethyl ether (10 ml).The solid was dissolved in water and treated with HCl (aq, 2M) to pH 3.The precipitate was filtered, washed with water, dried under vacuum toprovide pure 74 as a white solid (1.55 g, 6.72 mmol, 82%). ¹H NMR (400MHz, DMSO-d₆) δ 3.91 (3H, s), 8.02 (2H, dd, J 8.4 Hz), 8.22 (2H, dd, J8.4 Hz), 8.66 (2H, dd, J 10.4 Hz).

4-hydroxy-3-(6-(methoxycarbonyl)-2-naphthamido)benzoic acid (75). Asolution of 3-amino-4-hydroxybenzoic acid (0.142 g, 0.927 mmol) and theacyl chloride prepared from acid 74 (in 84% yield) (0.259 g, 1.019 mmol)in DCM (5 ml) and pyridine (6 ml) was stirred at room temperature forunder at for 24 h. The solvent was removed under reduced pressure. HCl(aq, 4 M, 6 ml) was added and the precipitate was filtered, washed withwater (10 ml) and dried under vacuum. Trituration with methanol (15 ml)and acetone (10 ml) afforded 75 as an off-white solid (0.084 g, 0.230mml, 25%). ¹H NMR (400 MHz, DMSO-d₆) δ 3.92 (3H, s), 7.00 (1H, d, J 8.4Hz), 7.67 (1H, d, J 8.8 Hz), 8.04-8.10 (2H, m), 8.16-8.27 (2H, m), 8.33(1H, s), 8.70-8.66 (2H, m), 9.80 (1H, s), 10.55 (1H, bs). ¹³C NMR (100MHz, DMSO-d₆) δ 53.09, 116.18, 122.59, 125.94, 126.45, 126.26, 126.80,128.45, 128.48, 129.19, 130.35, 130.40, 130.93, 134.13, 134.66, 134.94,154.61, 165.85, 166.84, 167.73. ν_(max) (solid)/(cm⁻¹) 3059, 1716, 1666,1592, 1541, 1283, 1247, 1132, 759.

Example 12—Effects of Compounds on Activation of STATs, Shc, and Erks

The following experiments and results are described and shown inSiddiquee, K. et al., PNAS USA, 2007, May, 104(18):7391-7396, Epub 2007Apr. 26, which is incorporated by reference herein in its entirety.Nuclear extracts containing activated Stat1, Stat3, and Stat5 proteinswere pre-incubated with increasing concentrations of compounds for 30minutes at room temperature prior to incubation with radiolabeled hSIEprobe that binds Stat1 and Stat3 or MGFe probe that binds Stat1 andStat5 and subjecting to EMSA analysis. Cell lysates containing activatedStat3 were pre-incubated with NSC 74859 in the presence and absence ofincreasing amount of cell lysates containing inactive Stat3 protein(monomer) prior to incubation with radiolabeled hSIE probe andsubjecting to EMSA analysis. Nuclear extract preparations fromv-Src-transformed NIH3T3/v-Src fibroblasts treated for the timesindicated in FIG. 2 of Siddiquee, K et al. (2007) or human breast cancerMDA-MB-231 and MDA-MB-468 treated for 48 hours with 100 μM NSC 74859were incubated with radiolabeled hSIE probe and subjected to EMSAanalysis. SDS-PAGE and Western blot analysis of whole-cell lysates fromNIH3T3/v-Src fibroblasts treated with or without NSC 74859 (100 μM, 48hours) for pTyr705Stat3 or Stat3. SDS-PAGE and Western blot analysis ofcell lysates prepared from NIH3T3/v-Src or EGF-stimulated NIH3T3/hEGFRwas carried out using antibodies against pShc, pErk1/2 (pp42/pp44), Erk,or β-actin, or antiphosphotyrosine antibody, clone 4G10. Controlsincluded nuclear extracts untreated with compounds, and nuclear extractsor lysates prepared from untreated cells.

Virtual high throughput screening relied upon computational modeling ofthe native pTyr peptide sequence (APY*LKT; SEQ ID NO:1) from one Stat3monomer bound within the SH2 domain of the second Stat3 monomer, asobserved for the Stat3 dimer protein bound to DNA (Becker, S. et al.Nature, 1998, 394:145-151). For the docking studies, DNA was removed andonly one of the two monomers was employed. The approach was used toevaluate the NCI Diversity Set and Plated Set chemical libraries.Three-dimensional structures for compounds from the NCI Diversity Setand NCI Plated Set were downloaded from NCI's DTP website (presented inSiddiquee, K. et al. (2007)), processed with LigPrep (36; available fromSchrödinger, L.L.C.) to produce 2,392 3D structures for the DiversitySet and 150,829 3D structures for the Plated Set. These structures weredocked using GLIDE 2.7 in SP (Standard Precision) mode into the pTyrpeptide binding site within the SH2 domain of the monomer employed. Thebest scoring compounds (74 compounds selected via visual inspection fromthe top 100 compounds with the best score from the Diversity Set and thetop 200 compounds from the Plated Set that then were filtered by MW(<700) and computed logs (>−10) to yield 122 compounds) were selectedfor evaluation in in vitro Stat3 DNA-binding assay. Nuclear extractscontaining activated STATs were incubated for 30 minutes with or withoutincreasing concentrations of compounds prior to incubation withradiolabeled hSIE probe that binds to Stat1 and Stat3 or MGFe probe thatbinds Stat1 and Stat5 and subjected to EMSA analysis. Results for arepresentative number of compounds show differential inhibition of DNAbinding activity of Stat3 following preincubation of nuclear extractswith compounds (Table 4) (data not shown for the remainder of the 194compounds evaluated in the DNA-binding assay). Potent inhibition ofStat3 DNA-binding activity by the NCI Plated Set compounds, NSC 42067,NSC 59263, NSC 74859, and NSC 75912, was observed. Other notable potentinhibitors of Stat3 DNA-binding activity from the Plated Set include NSC11421, NSC 91529, and NSC 263435 (Table 4). A few compounds with weakinhibitory activity were also identified from the NCI Diversity Set(data not shown). To determine selectivity against other STAT familymembers, selected active compounds were evaluated in in vitroDNA-binding assay of the three STAT proteins, Stat1, Stat3, and Stat5,in nuclear extracts prepared from EGF-stimulated NIH3T3/hEGFRfibroblasts that activates all three STATs. EMSA analyses of theDNA-binding activities of STAT proteins show that of the representativecompounds, NSC 74859 preferentially inhibits Stat3 over Stat1 or Stat5DNA-binding activity (Table 4), while NSC 42067 and NSC 59263preferentially inhibit Stat3 and Stat5 over that of Stat1 (Table 4). Inrecognition of the role of constitutive Stat5 in hematological and othercancers, the inhibition of Stat5 DNA-binding activity could haveclinical implications. Where Stat1 DNA-binding activity was inhibited,as with NSC 42067 and NSC 59263, it occurred at concentrations 3-4 timeshigher than concentrations that inhibited Stat3 or Stat5 (Table 4). Incontrast, NSC 75912 preferentially inhibits Stat1 over Stat3 or Stat5(Table 4). For the remaining compounds, no appreciable pattern ofspecificity of inhibition of the STAT family members was observed (Table4 and data not shown). These studies identify NSC 42067, NSC 59263, andNSC 74859 from the NCI chemical libraries as binders within the SH2domain of Stat3 and which potently inhibit Stat3 DNA-binding activity.The best of these compounds is NSC 74859 (re-synthesized as a puresample named BG2065p or S3I-201).

TABLE 4 IC₅₀ values for the inhibition of STATs DNA-binding activity invitro IC₅₀ (μM) NSC# Stat3:3 Stat1:3 Stat1:1 Stat5:5 11421 80 145 105221 24056 245 256 267 235 26685 85 68 95 125 42067 65 86 197 88 51926292 300 300 >300 59263 72 89 226 69 71906 295 258 225 285 74859 86160 >300 166 75912 160 104 72 >300 75914 276 263 113 >30087849 >300 >300 >300 >300 90438 >300 >300 >300 >300 91529 95 85 155 85101595 275 >300 >300 288 117907 202 292 >300 >300 205965 225 256 253 255216360 220 290 >300 >300 263435 25 40 50 25 645885 245 250 205 250647608 295 >300 >300 >300

Example 13—Inhibition of STAT3 Dimerization

Table 5 shows half maximal inhibitory concentration (IC₅₀) forinhibition of STAT3 dimerization, as measured by the EMSA asay (Yu, C.L. et al. Science, 1995, 269:81-83; Garcia, R. et al. Oncogene, 2001,20:2499-2513; Turkson, J. et al. Mol. Cell. Biol., 1998, 18:2545-2552).

TABLE 5 Inhibition of STAT3 dimerization IC₅₀ for the inhibition ofCompound # Compound Identifier FIG. STAT3 dimerization 1 NSC-74859 7<500 μM 2 BG2066 28 >500 μM 6 BG3006A 31 >500 μM 7 BG3006B 32 >500 μM 8BG3006D 33 >500 μM 11 BG2069-1 23 >500 μM 15 BG2074 29 >500 μM 19 BG300430 >500 μM 20 BG3009 34 >500 μM 21 HL2-006-1 13 >500 μM 22 HL2-006-214 >500 μM 23 HL2-006-3 15 >500 μM 24 HL2-006-4 16 <500 μM 25 HL2-006-517 <500 μM 27 HL2-011-2 19 >500 μM 28 HL2-011-5 22 >500 μM 29 HL2-011-624 >500 μM 30 HL2-011-6 25 >500 μM 31 HL2-005 26 >500 μM 32 HL2-00327 >500 μM 36 RPM381 35 <500 μM 38 RPM384 35 <500 μM 39 RPM385 35 <500μM 55 RPM405 36 >500 μM 59 RPM411 36 >500 μM 37 RPM407 37 >500 μM 40RPM412 37 >500 μM 56 RPM408 38 >500 μM 60 RPM410 38 >500 μM 57 RPM41539 >500 μM 61 RPM416 39 >500 μM 58 RPM418 40 >500 μM 62 RPM418-A 40 >500μM 65 RPM427 41 >500 μM 45 RPM431 42 >500 μM 73 RPM431 43 >500 μM 70RPM444 44 >500 μM 72 RPM448 44 >500 μM 69 RPM445 45 >500 μM 71 RPM44745 >500 μM 68 RPM452 46 >500 μM 75 RPM202 47 <500 μM

All patents, patent applications, provisional applications, andpublications referred to or cited herein, supra or infra, areincorporated by reference in their entirety, including all figures andtables, to the extent they are not inconsistent with the explicitteachings of this specification.

We claim:
 1. A method of treating a cancer having aberrant SignalTransducer and Activator of Transcription 3 (Stat3) activity in asubject, comprising administering an effective amount of digallic acid,or a pharmaceutically acceptable salt thereof, to the subject having thecancer.
 2. The method of claim 1, wherein the cancer is breast cancer.3. The method of claim 1, wherein the digallic acid, or pharmaceuticallyacceptable salt thereof, is administered locally at the site of a tumor.4. The method of claim 1, wherein the subject is human or a non-humanmammal.
 5. The method of claim 1, further comprising identifying thesubject as one suffering from the cancer.
 6. The method of claim 1,wherein the cancer is ovarian cancer, cervical cancer, lung cancer,esophageal cancer, colon cancer, bladder cancer, stomach cancer,pancreatic cancer, prostate cancer, leukemia, or melanoma.